PRODUCTION AND USE OF PLANT DEGRADING MATERIALS

Abstract
Disclosed herein are materials useful for degrading plant biomass material. In exemplary embodiments, the plant material comprises one or more enzymes that are expressed in plants and/or bacteria. Specifically exemplified herein are plant degrading enzymes expressed in chloroplasts. The chloroplast expressed enzymes may be provided as cocktails for use in conjunction with conventional methods of converting biomass into biofuels, such as cellulosic ethanol.
Description
BACKGROUND

The composition and diversity of biomass for producing cellulosic ethanol requires multiple enzyme systems, in very high concentrations, to release constituent sugars from which cellulosic ethanol is made. All currently available enzymes for cellulosic ethanol are produced in expensive fermentation systems and are purified in a process very similar to biopharmaceuticals like insulin. Therefore, reagent grade enzymes for ethanol production are extremely expensive. For example, B-glucosidase, pectolyase and cellulase are currently sold by Novozyme through the Sigma catalog for $124,000, $412,000 and $40,490 per kg, respectively. These enzymes are sold as formulations to bio-refineries without disclosing the actual enzyme components. Therefore, the actual cost for each enzyme, sold in bulk quantities, is not publicly available. Most industrial estimates for enzymes to produce cellulosic ethanol are in the $2 to $3 per gallon range, making large scale use cost prohibitive. Current capacity of fermentation systems will also be a major limitation. With increase in demand for enzymes and limited production capacity, the enzyme cost is likely to increase further.


A major limitation for the conversion of this biomass to ethanol is the high cost and large quantities of enzymes required for hydrolysis. B-glucosidase, pectolyase and cellulase are currently sold by Novozyme or other industries through the Sigma catalog for $124,000, $412,000 and $40,490 per kg, respectively. Therefore, the US DOE has long identified the cost of enzymes and their high loading levels required for most lingo-cellulosic feedstocks as one of the major barriers to cellulosic ethanol production. Currently, all commercially-available enzymes are produced through a fermentation process. Unfortunately, the building and maintenance of the fermentation production process is very expensive, costing $500M-$900M in upfront investment. No viable alternative to fermentation technology has yet emerged for mass-producing critical, yet prohibitively expensive industrial enzymes. This void in the marketplace for an alternative process is addressed directly by this proposal.





BRIEF DESCRIPTION OF DRAWINGS


FIG. 1 (a) Schematic representation of the chloroplast 16S trnI/trnA region. Transgenes were inserted at the trnI/trnA spacer region in the tobacco chloroplast genome. (b) Schematic representation of the chloroplast transformation vectors. The gene of interest (GOI) is celD, celO, pelA, pelB, pelD, cutinase, lipY, egl1, egI, swo1, xyn2, axe1 or bgl1. Prrn, rRNA operon promoter; aadA, aminoglycoside 3′-adenylytransferase gene confers resistance to spectinomycin; 5′ UTR, promoter and 5′ untranslated region of psbA gene; 3′ UTR, 3′ untranslated region of psbA gene. (c) Evaluation of transgene integration and homoplasmy by Southern blot of pelB, (d) pelD and (e) celD transplastomic lines hybridized with the flanking sequence probe (1, untransformed; 2 to 4, transplastomic lines). (f) Phenotypes of untransformed (UT) and transplastomic lines grown in green house showing normal growth.



FIG. 2 Western blot analysis and quantitation of transplastomic lines. Western blot of transplastomic lines expressing (a) PelB or (b) PelD. UT: untransformed, mature leaves harvested at 10 AM, 2 PM, 6 PM and 10 PM; 5 ng, 10 ng and 25 ng: PelA purified protein, young, mature and old leaves. (c) Enzyme units of PelB and PelD (c) or CelD (d) from one g or 100 mg leaf of different age or harvesting time.



FIG. 3 Effect of substrate, pH, temperature and cofactors on cpPelB, rPelB, cpPelD, rPelD, rCelD and cpCelD enzyme activity. (a) Effect of increasing PGA concentration on pectate lyases activity.(b) Effect of pH on pectate lyases activity in the absence of CaCl2 and (c) in the presence of CaCl2. The buffers used were following: 50 mM phosphate buffer (pH 6-7), Tris-HCl buffer (pH 8), glycine/NaOH buffer (pH 9) and CAPS buffer (pH 10.0) with 4 μg of TSP of PelB and PelD from both plant and E. coli. The optimal pH was determined at 40° C. using 2.5 mg/ml PGA as a substrate. (d) Effect of temperature (30-70° C.) on enzyme activity at pH 8.0 in the absence of CaCl2 and (e) in the presence of CaCl2. (f) Optimization of pH for cpCelD and rCelD activity using CMC (2%) at 60° C. for 30 minutes. Relative activity (%) was measured with reference to maximum activity obtained with 25 μg/ml for cpCelD and 10 μg/ml for rCelD (g) Effect of increasing temperature on relative activity of cpCelD and rCelD using CMC (2%) for 30 minutes at pH 6.0. Relative activity (%) was measured with reference to maximum activity obtained with 25 μg/ml for cpCelD and 10 μg/ml for rCelD (h) Enhancement of cpCelD (25 μg TSP/ml reaction) activity using 10 mM CaCl2 and 20 μg/ml BSA individually or in combination with 50 mM sodium acetate during the prolonged enzymatic hydrolysis. The hydrolysis was carried out up to 36 hours at 60° C., pH 6.0 in the presence of CMC (2%)



FIG. 4
E. coli vs chloroplast derived enzymes at different protein concentrations of crude extracts. (a) Enzyme kinetics of cpCelD and rCelD using carboxymethyl cellulose (2%) substrate. The reaction mixture contained increasing concentration of cpCelD and rCelD TSP (μg/ml) with 10 mM CaCl2 and 50 mM sodium acetate buffer, pH 6.0. Enzyme hydrolysis was carried out for 30 minutes at 60° C. Figure inset shows enzyme kinetics saturation point for cpCelD TSP amount (μg/ml) towards CMC (2%). Eppendorf tubes with reaction mixture shown in inset represents, 1 untransformed plant, 2 and 3 rCelD and cpCelD 10 μg TSP. (b) Effect of cpPelB, cpPelD, rPelB, and rPelD on hydrolysis of 5.0 mg/ml sodium polygalacturonate substrate. The reaction mixture contained increasing concentration of cpPelB, cpPelD, rPelB, and rPelD (μg/ml) in 20 mM Tris-HCl buffer (pH 8.0). Sodium polygalacturonate (Sigma) was measured using DNS method and measured from the D-galacturonic acid standard graph. Enzyme hydrolysis was carried out for 2 hour at 40° C. on rotary shaker at 150 rpm.



FIG. 5 Enzyme cocktails for filter paper, processed wood and citrus peel. (a) Filter paper activity was determined using Whatman No. 1 filter paper strip (50 mg/ml assay) at pH 5.5 and 50° C. Different combinations of crude extracts containing rEg1 (100 μg/ml), rBgl1 (200 μg/ml), rSwo1 (120 μg/ml), rCelO (100 μg/ml) and cpCelD (100 μg/ml) were used in the cocktail. The samples were incubated with 10 mM CaCl2, 20 μg BSA in a rotary shaker at 150 rpm for 24 hours. (b) Hydrolysis of processed wood sample (200 mg/5 ml reaction) was done by using a cocktail of crude extracts of cpPelB (250 μg/ml), cpPelD (250 μg/ml) (at pH 8.0), cpCelD (200 μg/ml), cpXyn2 (200 μg/ml), rEg1 (100 μg/ml), rBgl1 (200 μg/ml), rSwo1 (120 μg/ml), rCelO (100 μg/ml), rAxe1 (100 μg/ml), rPelA (200 μg/ml), rCutinase (50 μg/ml), rLipY (100 μg/ml). The reaction mixture containing 10 mM CaCl2, 20 μg/ml BSA was incubated for 36 hours in a rotary shaker at 150 rpm; pH (5.5-8.0) and temperature (40° C.-50° C.) were adjusted based on optimal conditions. Endpoint analysis of release of glucose equivalents was determined using DNS method. (c) Hydrolysis of Valencia orange peel (200 mg/5 ml reaction) was done using a cocktail of crude extracts of cpPelB (250 μg/ml), cpPelD (250 μg/ml) cpCelD (100 μg/ml) and cpXyn2 (100 μg/ml), reg1 (100 μg/ml), rBgl1 (200 μg/ml), rSwo1 (120 μg/ml), rCelO (100 μg/ml) and cpCelD (100 μg/ml), rAxe2 (100 μg/ml), rCutinase (50 μg/ml), rLipY (100 μg/ml), rPelA (200 μg/ml). End product analysis of reducing sugar was determined using DNS reagent31 and D-glucose and D-galacturonic acid as standards. Ampicillin and kanamycin 100 μg/ml was added to prevent any microbial growth during hydrolysis. The samples were incubated with 10 mM CaCl2, 20 μg/ml BSA in a rotary shaker at 150 rpm for 24 hours; pH (5.5-8.0) and temperature (40° C.-50° C.) were adjusted based on optimal conditions. In all experiments control assays contained substrate without enzyme or enzyme without substrate. All experiments and assays were carried out in triplicate.



FIG. 6 Generation of transplastomic tobacco commercial cultivars (A) Rooting of CelD LAMD shoot (B) CelD LAMD transplastomic plants growing in the green house (C) CelD LAMD transplastomic plants showing normal flowering (D) CelD TN90 primary transformant (E) Second round of regeneration for CelD TN90 (F) Rooting of PelB TN90 (G-I) First, second and third round of regeneration for PelB LAMD (J) Rooting of PelD LAMD shoot (K) PelD LAMD transplastomic plants growing in green house (L) PelD LAMD transplastomic plant showing normal flowering (M) Rooting of eg1 LAMD shoot (N) eg1 LAMD transplastomic plant growing in pots.



FIG. 7 Confirmation of homoplasmy by southern blots using tobacco flanking probe (A) CelD LAMD (B) PelB LAMD (C) PelB TN90 (D) PelD LAMD and (E) eg1 LAMD (UT: Untransformed plant; Numbers: Transplastomic lines and B: Blank).



FIG. 8 Enzymatic activity of pectate lyase B and D in Petit Havana, TN90 and LAMD tobacco cultivars (A) PelB (B) PelD Note: The leaf material used for the analysis of enzyme activity for TN90 and LAMD tobacco cultivars were harvested from in vitro plants, whereas the leaf material for Petit Havana is from the green house. Since the transgene is controlled by psbA, with light and developmental regulatory elements, expression levels in commercial cultivars are expected to be higher when transferred to the green house.



FIG. 9 shows sequence information of a few examples of plant degrading compound genes.





GENERAL DESCRIPTION

Certain embodiments of the invention address the major problems discussed above by producing all required enzymes in plants or bacteria or a combination of both, thereby dramatically alleviating the cost of fermentation and purification. According to one embodiment, the invention pertains to a method of degrading a plant biomass sample so as to release fermentable sugars therein. The method involves obtaining a plant degrading cocktail comprising at least two cell extracts, each cell extract having an active plant degrading compound that was recombinantly expressed in cells from which each said cell extract is derived. The at least two cell extracts are either plant extracts or bacterial extracts, or a combination of both. The plant degrading cocktail is admixed with the biomass sample to release fermentable sugars. In a more specific embodiment, the plant degrading cocktail includes cell extracts that include plant degrading enzymes such as cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, lipase, maltogenic alpha-amylase, pectolyase, or compounds that facilitate access of such enzymes, or so called, accessory plant degrading compounds, including but not limited to cutinase or expansin (e.g. swollenin). The inventors have found that such accessory plant degrading compounds serve to facilitate the action of plant degrading enzymes which synergistically elevates the amount of fermentable sugars produced for any given biomass sample.


Plant cell extracts will in most cases include an amount of ribulose-1,5-bisphosphate carboxylase_oxygenase (rubisco) from the plant cells from the plant cell extracts are derived. Rubisco is the most prevalent enzyme on this planet, accounting for 30-50% of total soluble protein in the chloroplast; it fixes carbon dioxide, but oxygenase activity severely limits photosynthesis and crop productivity (Ogren, W. L. (2003) Photosynth. Res. 76, 53-63, 2. Spreitzer, R. J. & Salvucci, M. E. (2002) Annu. Rev. Plant Biol. 53, 449-475). Rubisco consists of eight large subunits (LSUs) and eight small subunits (SSUs). The SSU is imported from the cytosol, and both subunits undergo several posttranslational modifications before assembly into functional holoenzyme (Houtz, R. L. & Portis, A. R. (2003) Arch. Biochem. Biophys. 414, 150-158.).


The use of genetic transformation of plants is generally not considered a viable alternative to the conventional fermentation processes for producing plant degrading enzymes in light of the fact that the expressed proteins would be deleterious to plant life and growth. The inventors have endeavored to devise a method of expressing plant degrading enzymes in such a way that does not disrupt the plant cell. It is the inventors' belief that the present invention is the first demonstration of viable plant degrading enzyme expression in plants. The inventors have realized that the expression of many plant degrading enzymes can be expressed in chloroplasts without adverse effects on the plant. The chloroplasts appear to insulate the plant cell from damage from the enzymes. Though expression in chloroplasts is exemplified herein, unless specifically stated, embodiments of the present invention should not be construed to be limited to chloroplast expression of plant degrading enzymes. Certain embodiments related to a combination of plant and bacterial extracts from cells engineered to recombinantly express plant degrading compound(s).


The term “recombinantly expressed” as used herein refers to production of a polypeptide from a polynucleotide that is heterologous to the cell in which the polynucleotide has been transfected. Recombinant expression may result from heterologous polynucleotides that are stably transformed in the genome of the cell, or genome of the cell organelle, or which are merely present in the cell via a transfection event.


The term chloroplast is interpreted broadly to cover all plastids, including proplastids, etioplasts, mature chloroplasts, and chromoplasts.


A comprehensive cellulase system consists of endoglucanases, cellobiohydrolases and beta glucosidases. The cellobiohydrolases and endoglucanases work synergistically to degrade the cellulose into cellobiose, which is then hydrolysed to glucose by the beta glucosidases. Cellobiase, or beta-glucosidase, activity is responsible for the formation of glucose from cellobiose and plays an important role in cellulose degradation by relieving the end product (cellobiose) inhibition. Gene sequences for most of these enzymes, from different microorganisms, have been deposited in public data bases.


Pectins, or pectic substances, are collective names for a mixture of heterogeneous, branched and highly hydrated polysaccharides present as one of the major components in plant cell walls. These polysaccharides comprise mostly neutral sugars, such as arabinan, galactan, andarabino galactan. Activepectolytic enzyme preparations have the following enzymes: Two alpha-L-rhamnohydrolases, polygalacturonase, pectin methylesterase, endo-pectate lyase (pectintranseliminase), pectin lyase and small percent of xylanase. Nucleotide sequence for pectin degrading enzymes, xylanases, cellulases are available in public data bases. See Example 9 herein for discussion on sequences.


The inventor has realized that enzyme requirements are very different for each type of biomass used in cellulosic ethanol production. Accordingly, certain embodiments of the present invention relate to cocktails of enzymes obtained from plant expression and/or bacteria expression that the inventors have developed to be particularly effective for the targeted biomass material.


Biomass sources that can be degraded for ethanol production in accordance with the teachings herein, include, but are not limited, to grains such as corn, wheat, rye, barley and the grain residues obtained therefrom (primarily leftover material such as stalks, leaves and husks), sugar beet, sugar cane, grasses such as switchgrass and Miscanthus, woods such as poplar trees, eucalyptus, willow, sweetgum and black locust, among others, and forestry wastes (such as chips and sawdust from lumber mills). Other biomasses may include, but are not limited to, fruits including citrus, and the waste residues therefrom, such as citrus peel.


Throughout this document, tobacco is referred to as an exemplary plant for expressing plant degrading enzymes. However, unless specifically stated, embodiments of the invention should not be construed to be limited to expression in tobacco. The teachings of gene expression taught herein can be applied to a wide variety of plants, including but not limited to tobacco; lettuce, spinach; sunflower; leguminous crops such as soybean, beans, peas, and alfalfa; tomato; potato; carrot; sugarbeet; cruciferous crops; fibre crops such as cotton; horticultural crops such as gerbera and chrysanthemum; oilseed rape; and linseed.


In one embodiment, genes from Aspergillusniger, Aspergillus aculeatus, Trichodermareesei andor Clostridium thermocellum encoding different classes of enzymes are isolated using gene specific primers. In order to express different classes of genes in a chloroplast of interest, the following strategies are used. The chloroplast vector contains flanking sequences from the chloroplast genome to facilitate homologous recombination. In one embodiment, foreign genes are integrated individually into the spacer region of chloroplast genome. The coding sequence of different enzymes can be regulated by appropriate regulatory sequences. Recombinant plasmids will be bombarded into tobacco to obtain transplastomic plants.


In a specific embodiment, powdered tobacco leaves are used as enzyme sources for commercial evaluation of ethanol production from a biomass source. In a more specific embodiment, the biomass source is a grain such as corn, a grass, or is citrus waste.


For chloroplasts that are transformed, it has been realized that obtaining homplasmy with respect to the transgenic chloroplasts is desired. The transgene integration and homoplasmy is confirmed by PCR and Southern blot analysis, respectively. Expression of the transgenes is confirmed by western blot analysis and quantified by ELISA. The protein extract from transplastomic tobacco plants is tested for its ability to degrade citrus waste biomass. Based on the results, more enzyme classes are added to increase the breakdown of plant biomass to sugars for fermentation to ethanol. Each tobacco plant, engineered to produce an enzyme, will be able produce a million seeds, to facilitate scale up to 100 acres, if needed. Homogenized plant material, such as powdered tobacco leaves or plant extracts, or purified enzymes from plant material are used as the enzyme source for commercial evaluation of ethanol production from citrus waste.


While current methods involve placing a foreign gene in the plant cell nucleus, CT transforms the genome of the approximately 100 chloroplasts that are within each tobacco plant cell. Each tobacco plant chloroplast contains about 100 copies of the chloroplast's genetic material, so the amount of protein (in this case, enzyme proteins used to break down biomass into sugar for ethanol production) is increased exponentially. This is the primary reason why massive volume production of cell wall degrading enzymes for cellulosic ethanol production is so cost effective. Secondly, tobacco has large volume biomass (40 metric tons of leaves per acre) and it can be harvested multiple times during a given growing season in Florida. And, as previously mentioned, it is easy to plant 100 acres from a single tobacco plant. Lastly, because chloroplasts are inherited maternal, they are not functional in the tobacco plant's pollen.


According to one embodiment, the invention pertains to a method of degrading a plant biomass sample to release fermentable sugars. The method includes obtaining a plant degrading cocktail having at least one chloroplast genome or genome segment having a heterologous gene that encodes cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase or pectolyase, or a combination thereof, and wherein said plant material has cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase or expansin, or a combination thereof, that has been expressed in a plant from which said plant material is derived; and admixing said plant degrading material with said biomass sample. In a more specific embodiment, the enzyme or combination of enzymes pertains to more than 0.1 percent of the total protein in the plant material.


According to another embodiment, the invention pertains to a method of producing a plant biomass degrading material sufficient to release fermentable sugars, the method including producing at least one plant comprising chloroplasts that express cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase, or expansin, or a combination thereof; harvesting said plant; and processing said plant to produce an enzyme source suitable for mixing with and degrading a biomass sample. In a specific embodiment the plant is tobacco; lettuce, spinach; sunflower; fibre crops such as cotton; horticultural crops such as gerbera and chrysanthemum; leguminous crops such as soybean, beans, peas, and alfalfa; tomato; potato; sugarbeet; cruciferous crops, including oilseed rape; and linseed. In a more specific embodiment, plant is tobacco.


One aspect of the invention is to provide an abundant inexpensive source of enzyme for degrading biomass. Accordingly, the plant or bacterial material in which plant degrading compounds have been expressed may be processed by drying and powderizing the plant or a portion thereof. In another embodiment, crude liquid extracts are produced from the plant and/or bacterial material. In alternative embodiments, the plant degrading material may be enzymes that have been purified fully or partially from the plant and/or bacteria in which they are expressed. However, providing the plant degrading material as a dry form or as crude extract of the plant and/or bacterial material avoids the need for time-consuming and potentially expensive purification steps. In this way, the plant material has a longer shelf life and may easily be mixed with the plant biomass sample according to conventional plant degrading and fermenting processes.


In one specific embodiment, the method of producing a plant entails producing a first plant with chloroplasts transformed to express a first enzyme and a second plant with chloroplasts transformed to express a second enzyme. Plant material from both first and second plants may be combined to produce a plant degrading sample that includes more than one plant degrading enzyme. In a more specific embodiment, the invention pertains to a method of producing a plant that entails at least two of the following: producing a first plant comprising chloroplasts that express cellulase, producing a second plant comprising chloroplasts that express lignanse, producing a third plant comprising chloroplasts that express beta-glucosidase; producing a fourth plant comprising chloroplasts that express hemicellulase; producing a fifth plant comprising chloroplasts that express xylanase; producing a sixth plant comprising chloroplasts that express alpha-amylase; producing a seventh plant comprising chloroplasts that express amyloglucosidase; producing an eighth plant comprising chloroplasts that express pectate lyase; producing a ninth plant comprising chloroplasts that express cutinase; producing a tenth plant comprising chloroplasts that express lipase; producing an eleventh plant comprising chloroplasts that express maltogenic alpha amylase, producing a twelfth plant comprising chloroplasts that express pectolyase and/or a thirteenth plant comprising chloroplasts that express expansin (e.g. swollenin).


As alluded to above, the inventors have recognized that according to certain embodiments, plant derived enzymes are augmented with plant degrading enzymes recombinantly expressed in bacteria. Thus, a plant degrading cocktail may include enzymes recombinantly expressed in plants and enzymes that are recombinantly expressed in bacteria, such as but not limited to E. coli.


According to a further embodiment, the invention pertains to a plant material useful for degrading a plant biomass, the material including at least one chloroplast genome or genome segment having a heterologous gene that encodes cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase, or expansin, or a combination thereof; and wherein said plant material comprises cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase, or expansin, or a combination thereof. In a more specific embodiment, the plant material includes at least two of the following: a first chloroplast genome or genome segment having a heterologous gene that encodes cellulase, a second chloroplast genome or genome segment having a heterologous gene that encodes lignanse, a third chloroplast genome or genome segment having a heterologous gene that encodes beta-glucosidase; a fourth chloroplast genome or genome segment having a heterologous gene that encodes hemicellulase; a fifth chloroplast genome or genome segment having a heterologous gene that encodes xylanase; a sixth chloroplast genome or genome segment having a heterologous gene that encodes alpha-amylase; a seventh chloroplast genome or genome segment having a heterologous gene that encodes amyloglucosidase; an eighth chloroplast genome or genome segment having a heterologous gene that encodes pectate lyase; a ninth plant chloroplast genome or genome segment having a heterologous gene that encodes cutinase; a tenth chloroplast genome or genome segment having a heterologous gene that encodes lipase; an eleventh chloroplast genome or genome segment that encodes maltogenic alpha-amylase, a twelfth chloroplast genome or genome segment having a heterologous gene that encodes pectolyase and a thirteenth chloroplast genome or genome segment having a heterologouse gene that encodes expansin (e.g. swollenin).


According to another embodiment, the invention pertains to a plant having a plant cell having a more than natural amount of cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase pectolyase or expansin, or a combination thereof, and wherein said plant material comprises cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, pectolyase, or expansin, or a combination thereof, active enzyme therein. In a more specific embodiment, the enzyme or combination of enzymes represents is more than 0.1 percent of the total protein of the cell. In an even more specific embodiment the enzyme or combination of enzymes represent more than 1.0 percent of the total protein in the cell.


In a further embodiment, the invention pertains to a plant derived composition comprising cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase and/or swollenin, and an amount of rubisco. In a specific embodiment, the rubisco to enzyme ratio ranges from 99:1 to 1:99.


In an additional embodiment, the invention pertains to commercial cultivars that recombinantly express one or more plant degrading compounds. Commercial cultivars of tobacco, such as, but not limited to, LAMD and TN90, produce substantially more leaf material than experimental cultivars. However, commercial cultivars are more sensitive to introduction of foreign genes. Surprisingly, the inventor, through significant trial and error, has been able to successfully transfect cells and induce expression of plant degrading compounds in commercial cultivars.


EXAMPLES
Example 1
Assembly of Chloroplast Expression Constructs

For the integration of transgenes, transcriptionally active spacer region between the trnI and trnA genes was used (FIG. 1a). PCR resulted in the amplification of various genes of interest (GOI) including endoglucanase (celD), exoglucanase (celO) from Clostridium thermocellum genomic DNA, lipase (lipY) from Mycobacterium tuberculosis genomic DNA, pectate lyases (pelA, pelB, pelD) and cutinase from Fusarium solani. Using a novel PCR based method, coding sequences of GOI including endoglucanases (egl1 and egI), swollenin (swo1 similar to expansins), xylanase (xyn2), acetyl xylan esterase (axe1) and beta glucosidase (bgl1) were cloned without introns (ranging from 1-5) from Trichoderma ressei genomic DNA. Tobacco chloroplast transformation vectors were made with each GOI (FIG. 1b). All chloroplast vectors included the 16S trnI/trnA flanking sequences for homologous recombination into the inverted repeat regions of the chloroplast genome and the aadA gene conferring resistance to spectinomycin. The aadA gene was driven by the constitutive rRNA operon promoter with GGAGG ribosome binding site. The GOI was driven by the psbA promoter and 5′ UTR in order to achieve high levels of expression. The 3′ UTR located at the 3′ end of the GOI conferred transcript stability.


Example 2
Generation and Characterization of Transplastomic Tobacco Expressing Pectate Lyases (PelB & PelD) and Endoglucanase (CelD)

Transplastomic tobacco plants were obtained as described previously28, 29. Southern blot analysis was performed to confirm site specific integration of the pLD-pelB, pLD-pelD and pLD-celD cassettes into the chloroplast genome and to determine homoplasmy. Digestion of total plant DNA with SmaI from untransformed and transplastomic lines generated a 4.014 kb fragment untransformed (UT) or 6.410 kb in pelB, 6.377 kb in pelD or 7.498 kb fragment in celD when hybridized with the [32P]-labeled trnI-trnA probe, confirming site specific integration of the transgenes into the spacer region between the trnI and trnA genes (FIG. 1c-e). Furthermore, the absence of a 4.014 kb fragment in the transplastomic lines confirmed that homoplasmy was achieved (within the levels of detection). The phenotypes of transplastomic lines appeared to be normal when compared to untransformed plants and were fertile (produced flowers, seeds FIG. 1f).


Immunoblots with antibodies raised against PelA showed that PelB and PelD are immunologically related to PelA30. Therefore, PelA antibody was used to detect the expression of PelB and PelD, although their affinity was variable in transplastomic lines. All transplastomic lines showed expression of PelB or PelD protein. Expression levels did not change significantly corresponding to their enzyme activity depending on the time of harvest even though the PelB and PelD are regulated by light (FIG. 2a,b). This may be because of variable affinity between antigen epitopes of PelB, PelD and PelA antibody. Enzyme concentration slightly changed with leaf age and decreased in older leaves (FIG. 2a,b).


Example 3
Quantification of Pectate Lyases (PelB, PelD), and Endoglucanase (CelD) at Different Harvesting Time and Leaf Age

The activity of the enzyme varied significantly depending on the developmental stages and time of leaf harvest. Maximum enzyme activity was observed in mature leaves of PelB, PelD and CelD, with reduced activity in older leaves (FIG. 2c,d). Mature leaves harvested at 6 PM showed maximum activity in both PelB and PelD whereas CelD showed maximum activity at 10 PM (FIG. 2c,d). This may be due to increased stability of endoglucanase against proteases in plant extracts. Activity of cpCelD did not significantly decrease in plant crude extracts stored at room temperature, for more than thirty days (data not shown).


CelD enzyme activity was calculated using DNS reagent31 according to the IUPAC protocol32. The specific activity of cpCelD using 2% CMC substrate was 493 units/mg total soluble protein (TSP) or 100 mg leaf tissue, in crude extracts prepared from mature leaves harvested at 10 PM. Using the glucose hexokinase assay, which is highly specific for glucose, the specific activity was 4.5 units/mg TSP and 6.28 units/mg TSP, when 5% avicel and sigmacell solution respectively was used as substrate (at pH 6.0, 60° C.). Commercial cellulase enzyme (Trichoderma reesei, EC 3.2.1.4, Sigma) gave 4.2 units/mg and 3.43 units/mg solid for the same substrate. FIGS. 2c and 2d show that approximately 26 units, 32 units and 4,930 units of PelB, PelD and CelD were obtained per gram fresh weight of mature leaves harvested at 6 PM or 10 PM. Thus, 2,048, 2,679 and 447,938 units of PelB, PelD and CelD can be harvested from each tobacco plant (experimental cultivar, Petit Havana). With 8,000 tobacco plants grown in one acre of land, 16, 21 and 3,584 million units of PelB, PelD or CelD can be obtained per single cutting (Table 1). Based on three cuttings of tobacco in one year, up to 49, 64 and 10,751 million units of PelB, PelD or CelD can be harvested each year. The commercial cultivar yields 40 metric tons biomass of fresh leaves as opposed to 2.2 tons in experimental cultivar Petit Havana. Therefore, the commercial cultivar is expected to give 18 fold higher yields than the experimental cultivar.


Example 4
Effect of pH & Temperature on Pectate Lyases (PelB & PelD) and Endoglucanase (CelD) Enzyme Activity

Both plant and E. coli extracts showed optimal activity at 2.5 mg/ml PGA (FIG. 3a). Therefore, all enzyme characterization studies were performed at this substrate concentration. Kinetic studies carried out by using 4 μg of TSP, with increasing concentration of PGA (0-2.5 mg), under standard assay conditions gave Km values of 0.39 and 1.19 μg/ml in chloroplast (cp) and E. coli (r) PelB respectively, whereas values for chloroplast and E. coli PelD were 0.50 and 1.29 μg/ml respectively. The Vmax values obtained were 2.75, 3.19, 2.75 and 3.14 units/mg for cpPelB, rPelB, cpPelD and rPelD, respectively (FIG. 3a).


The crude extract (4-5 μg TSP) from plant or E. coli was used to study the effect of pH and temperature on the activity of enzymes. The optimal temperature for the E. coli and chloroplast derived pectate lyase under the standard assay conditions was 40° C. and the optimal pH was 8.0. Plant derived pectate lyases showed a pH optimum of 6.0 in the presence of 1 mM CaCl2. The E. coli crude extracts showed an optimal pH of 8.0 irrespective of presence or absence of CaCl2 in the reaction (FIG. 3b,c). The temperature increase had minimal effect on the activity of plant derived pectate lyases, whereas the E. coli enzyme showed comparatively lower activity at higher temperatures (FIG. 3d,e). These differences in enzyme properties from two different hosts may be due to their folding. This possibility was supported by the observation that it was possible to detect the E. coli enzyme with HIS-tag antibody but not the chloroplast enzyme (data not shown). It is well known that foreign proteins form disulfide bonds in chloroplasts33-35 but not in E. coli when expressed in the cytoplasm. Both PelB and PelD enzymes have even number (12 or 14) cysteines that could form disulfide bonds30.


CpCelD activity with 2% CMC was measured at different pH and temperature. The cpCelD showed pH optima between pH 5.0 to pH 7.0 (FIG. 3f) whereas E. coli enzyme had a pH optimum of 6.5. Temperature optima was between 50-60° C. for E. coli and 50-70° C. for plant enzyme (FIG. 3g). Clostridium thermocellum CelD is structurally known to have affinity for CaCl2 ions and it also provided thermostability36. Even though 10 mM CaCl2 increased CelD activity in 2% CMC to 2 fold in E. coli crude extract, this was not apparent in chloroplast CelD crude extract during initial period of incubation. This may be due to optimum concentration of calcium ion present in plant cells. However, CaCl2 with 20 μg BSA yielded 5 fold increased activity at the end of 24 hour incubation for cpCelD crude extract (FIG. 3h).


Example 5

E. coli vs Chloroplast CelD, PelB & PelD


E. coli crude extract containing CelD enzyme showed decrease in enzyme activity when the reaction mixture contained more than 10 μg TSP, where as plant crude extract containing CelD released more reducing sugar with increasing protein concentration (FIG. 4a). Chloroplast expressed CelD activity was saturated (in 2% CMC) at 150 μg TSP (FIG. 4a inset) and there was no decrease in chloroplast CelD enzyme activity even up to 500 μg TSP as determined by end point assay. These results show that crude plant extracts containing cpCelD can be directly used for biomass degradation without any need for purification whereas E. coli extracts probably contain endoglucanase inhibitors. Similarly, at higher protein concentrations, E. coli expressed rPelB and rPelD showed reduced pectate lyase activity whereas cpPelB or cpPelD continued to increase activity even up to 600 μg TSP (FIG. 4b). There may be inhibitors of pectate lyase in E. coli extracts, which are not present in plant crude extracts. This finding is potentially of high practical significance because use of crude extracts eliminates the need for purification of enzymes. Large amounts of crude plant enzyme can be utilized in the cocktail as shown below without causing detrimental effect on enzyme activity, hydrolysis or yield of end products.


Example 6
Enzyme Cocktail for Hydrolysis of Filter Paper

Before evaluation of enzyme cocktails, activity of each enzyme was tested independently with an appropriate substrate. Chloroplast or E. coli expressed endoglucanase (cpCelD or rEg1) alone did not release any detectable glucose from filter paper but when mixed together up to 19% of total hydrolysis was observed (FIG. 5a, bar1). This could be due to different carbohydrate binding domains of endoglucanases towards filter paper. The synergistic activity was further enhanced up to 47 or 48% when the endoglucanases (cpCelD and rEg1) were mixed with swollenin (rSwo1) or beta-glucosidase (rBgl1, FIG. 5a, bar2&3). Addition of cellobiohydrolase (rCelO) to this cocktail doubled the hydrolysis of filter paper, releasing maximum amount of reducing sugar (FIG. 5a, bar4). This synergism observed was probably due to the exo-mode of action of cellobiohydrolase37 from reducing ends that were formed by random cuts in cellulose chains through endoglucanases (cpCelD and rEg1), along with the action of expansin and beta-glucosidase.


Example 7
Enzyme Cocktail for Hydrolysis of Processed Wood Sample

The enzyme cocktail that released highest glucose equivalents with filter paper (except rEg1) was tested on processed wood substrate. After 24 hour hydrolysis, 31% of total hydrolysis was observed with this cocktail (FIG. 5b, bar1). An enzyme cocktail of endoxylanase and acetyl xylan esterase showed 41% of total hydrolysis (FIG. 5b, bar2). When these two cocktails were combined together, the hydrolysis increased up to 88% (FIG. 5b, bar3). When processed wood substrate was first treated with pectate lyases, followed by the addition of the enzyme cocktail in bar 3, the overall hydrolysis was further enhanced, with release of up to 275 μg of glucose after 36 hour incubation (FIG. 5b, bar4). Addition of cutinase and lipase enzyme extracts (both with lipase activity) did not have significant effect on the release of fermentable sugars (data not shown). Novozyme 188 enzyme cocktail did not yield any detectable glucose equivalents from processed wood, whereas Celluclast 1.5 L yielded 10% more than the crude extract cocktail, with equivalent enzyme units based on CMC hydrolysis.


According to one embodiment, the invention pertains to a method for digesting a wood-based biomass sample comprising obtaining a plant material comprising endoxylanase or acetyl xylan esterase, or a combination thereof, that has been expressed in a plant from which all or a portion of said plant material is derived; and admixing said plant material with said wood based biomass sample. The method of this embodiment may further include admixing with the wood-based biomass sample, either prior to or contemporaneous to, admixture with the endoxylanase and/or acetyl xylan esterase plant material, a plant material comprising a pectate lyase that has been expressed in a plant from which all or a portion of said plant material is derived. The plant material may comprise rubisco.


Another embodiment pertains to a plant degrading enzyme cocktail useful in digesting a wood-based biomass sample comprising cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase, swollenin or pectate lyase, or a combination thereof expressed in a plant, optionally with an amount of rubisco.


Example 8
Enzyme Cocktail for Hydrolysis of Citrus Waste

The enzyme cocktail of endoglucanase (cpCelD), exoglucanase, swollenin and beta-glucosidase released up to 24% of total hydrolysis with citrus peel (FIG. 5c, bar1). When citrus peel was treated with pectate lyases (cpPelB, cpPelD and rPelA), hydrolysis was doubled (FIG. 5c, bar2). Pectate lyases contributed to 47% of total hydrolysis in this cocktail because of high pectin content (23%) in citrus peel41. Addition of endoxylanase, acetyl xylan esterase, cutinase and lipase to the both these cocktails released up to 360 μg/ml glucose equivalents from 100 mg ground citrus peel after 24 hour incubation period (FIG. 5c, bar3). Enzymes like cutinase and lipase may have hydrolyzed oil bodies present in the citrus peel, providing greater access to endoglucanase, endoxylanase and pectate lyases for efficient hydrolysis of citrus peel. Novozyme 188 enzyme cocktail yielded 11% more glucose equivalents with citrus peel, whereas Celluclast 1.5 L yielded 137% more than the crude extract cocktail with equivalent enzyme units based on CMC hydrolysis.


According to one embodiment, the invention pertains to a method for digesting a citrus biomass sample comprising obtaining a plant material comprising cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase or pectate lyase, or a combination thereof, that has been expressed in a plant from which all or a portion of said plant material is derived; and admixing said plant material with said citrus biomass sample.


Another embodiment pertains to a plant degrading enzyme cocktail useful in digesting a citrus biomass sample comprising cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase, swollenin or pectate lyase, or a combination thereof expressed in a plant, optionally with an amount of rubisco.


Methods Related to Examples 1-8

Isolation of Genes and Construction of Plastid Transformation Vectors


Genomic DNA of Clostridium thermocellum and Trichoderma reesei was obtained from ATCC and used as template for the amplification of different genes. Gene specific primers using a forward primer containing a NdeI site and a reverse primer containing a XbaI site for cloning in the pLD vector were designed for celD, celO and lipY genes. The mature region of cellulose genes celD (X04584) and celO (AJ275975) were amplified from genomic DNA of Clostridium thermocellum. LipY (NC000962) was amplified from genomic DNA of Mycobacterium tuberculosis. Overlapping primers were designed for the amplification of various exons of egl1 (M15665), egI (AB003694), swoI (AJ245918), axe1 (Z69256), xyn2 (X69574) and bgl1 (U09580) from genomic DNA of Trichoderma reesei using a novel method. Full length cDNA of these genes was amplified from different exons by a novel PCR based method using the forward of first exon and reverse of last exon containing a NdeI site and XbaI site respectively. Pectate lyase genes pelA, pelB & pelD from Fusarium solani with similar restriction sites were amplified using gene specific primers from pHILD2A, pHILD2B30 and pHILD2D45 respectively. A similar strategy was used to amplify cutinase gene46 from recombinant clone of Fusarium solani. All the full length amplified products were ligated to pCR Blunt II Topo vector (Invitrogen) and were subjected to DNA sequencing (Genewiz). Each gene cloned in Topo vector was digested with NdeI/XbaI and inserted into the pLD vector17, 47 to make the tobacco chloroplast expression vector.


Regeneration of Transplastomic Plants and Evaluation of Transgene Integration by PCR and Southern Blot



Nicotiana tabacum var. Petite Havana was grown aseptically on hormone-free Murashige and Skoog (MS) agar medium containing 30 g/l sucrose. Sterile young leaves from plants at the 4-6 leaf stages were bombarded using gold particles coated with vector pLD-PelB, pLD-PelD and pLD-CelD and transplastomic plants were regenerated as described previously28, 29. Plant genomic DNA was isolated using Qiagen DNeasy plant mini kit from leaves. PCR analysis was performed to confirm transgene integration into the inverted repeat regions of the chloroplast genome using two sets of primers 3P/3M and 5P/2M, respectively17. The PCR reaction was performed as described previously17, 29. Leaf from the PCR positive shoots were again cut into small pieces and transferred on RMOP (regeneration medium of plants) medium containing 500 mg/l spectinomycin for another round of selection and subsequently moved to MSO (MS salts without vitamins and growth hormones) medium containing 500 mg/l spectinomycin for another round of selection to generate homoplasmic lines. Southern blot analysis was performed to confirm homoplasmy according to lab protocol48. In brief, total plant genomic DNA (1-2 μg) isolated from leaves was digested with SmaI and hybridized with 32P α[dCTP] labeled chloroplast flanking sequence probe (0.81 kb) containing the trnI-trnA genes. Hybridization was performed by using Stratagene QUICK-HYB hybridization solution and protocol.


Immunoblot Analysis


Approximately 100 mg of leaf was ground in liquid nitrogen and used for immunoblot analysis as described previously48. Protein concentration was determined by Bradford protein assay reagent kit (Bio-Rad). Equal amounts of total soluble protein were separated by SDS-PAGE and transferred to nitrocellulose membrane. The transgenic protein expression was detected using polyclonal serum raised against PelA in rabbit.



E. coli Enzyme (Crude) Preparation



E. coli strain (XL-10 gold) harboring chloroplast expression vectors expressing rCelD, rEg1 (EC 3.2.1.4), rCelO (EC 3.2.1.91), rXyn2 (EC 3.2.1.8), rAxe1 (EC 3.1.1.72), rBgl1 (EC 3.2.1.21), rCutinase (EC 3.1.1.74), rLipY (lipase, EC 3.1.1.3), rPelA, rPelB, rPelD (EC 4.2.2.2) or rSwo1 was grown overnight at 37° C. Cells were harvested at 4° C. and sonicated four times with 30 s pulse in appropriate buffer (50 mM sodium acetate buffer with pH 5.5 for CelD, Eg1, CelO, Swo1, Xyn2, Axe1, Bgl1, 100 mM Tris-Cl with pH 7.0 for cutinase, lipase, PelA, PelB and PelD) containing protease inhibitor cocktail (Roche) and sodium azide (0.02%). Supernatant was collected after centrifugation at 16,000×g for 10 minutes and protein concentration was determined.


Enzyme Preparation from Tobacco Transplastomic Leaf Material


Fresh green leaves were collected and ground in liquid nitrogen. Total soluble protein was extracted in 50 mM sodium acetate buffer, pH 5.5 for cpCelD, cpXyn2 or 100 mM Tris-Cl buffer, pH 7.0 for PelD and PelB. All buffers contained protease inhibitor cocktail (Roche) and sodium azide (0.02%). Total soluble protein was filtered using 0.22 μm syringe filter, Protein concentration (mg/ml) in TSP was determined using Bradford method.


Enzyme Assays for Pectate Lyase B and Pectate Lyase D


Pectate lyases B and D were assayed spectrophotometrically by measuring the increase in A23530, 49, 50. Kinetics of the pectate lyase B and D were studied to optimize substrate concentration (0.0-2.5 mg) under identical protein and cofactor concentration. The reaction mixtures contained 1 ml of 50 mM Tris-HCl buffer (pH 8.0) with 1 mM CaCl2 (freshly prepared), 1 ml of 0.0-2.5 mg/ml sodium polygalacturonate (Sigma) and 0.5 ml of suitably diluted enzyme solution. Measurements were carried out at 40° C. One unit of enzyme was defined as the amount of enzyme which forms 1 μmol of product per min with a molar extinction coefficient of 4,600 μmol−1 cm−1. Kinetic studies were carried out in 50 mM Tris-HCl buffer, pH 8.0 at 40° C. Kinetic parameters (Km & Vmax) were calculated using non linear regression using Graphpad Prism 5.0. The initial slopes of each substrate concentration were calculated, where as the velocity (units/mg/min) was defined through the release of unsaturated galacturonic acid. The temperature optimization for pectate lyase B and D activity was carried out in 50 mM Tris-HCl buffer, pH 8.0 at different temperatures ranging from 30° C. to 70° C. In each case, the substrate was pre-incubated at the desired temperature for 5 min. In order to study the thermal stability of the enzyme, buffered enzyme samples were incubated for fixed time period at different temperatures.


The pH optimum of the pectate lyase B and D was measured at 40° C. using different buffers ranging from pH 6 to 10, with the same ionic strength. The stability of the crude extract of the enzyme was optimized by incubating the enzyme at the different pH. The influence of the cofactor CaCl2 on pectate lyase activity was studied by conducting the reactions in its presence and absence at different pH and temperature.


Enzyme Assay for CelD and Commercial Cocktail (Celluclast 1.5 L and Novozyme 188)


Cellulase enzyme activity of cpCelD was determined by incubating crude extract in 2% carboxylmethylcellulose, avicel and sigmacell (Sigma) as substrate according to IUPAC recommendations32 in 50 mM sodium acetate buffer pH 6.0 and incubated at 60° C. for 30 minutes for CMC and 2 hours for avicel and sigmacell. Enzyme units of commercial cocktails Celluclast 1.5 L and Novozyme 188 were determined using 2% CMC, under identical assay conditions. Reducing sugar amount was determined using 3,5-dinitrosalicylic acid31. D-glucose and D-galacturonic acid were used as standard to measure release of glucose equivalents and unsaturated galacturonic acid molecules. CMC (2%) was used in determining the pH and temperature activity profile of cpCelD. One unit of enzyme was defined as the amount of enzyme that released 1 μmole glucose equivalents per minute/ml. Cellulase unit calculation for avicel and sigmacell was based on glucose hexokinase method according to the manufacturer's protocol (Sigma).


Enzymatic Hydrolysis of Filter Paper, Processed Wood and Citrus Peel


Enzyme assays were carried out either with one enzyme component or as cocktail on filter paper, processed wood and orange peel and released reducing sugar was determined using DNS method. Orange peel prepared from Valencia orange (Citrus sinensis cv Valencia) fruit was air dried overnight and ground in liquid nitrogen. Ground Valencia orange peel and pretreated wood biomass were washed several times in distilled water until no reducing sugar was detected by DNS reagent as well as by glucose hexokinase method.


For enzymatic digestion, 50-200 mg of processed wood sample or ground orange peel was used. Crude extracts containing enzymes from E. coli and plants were used in the cocktail for hydrolysis. End product reducing sugar was determined using DNS reagent31 and D-glucose as standard. Ampicillin and kanamycin 100 μg/ml was added to prevent any microbial growth during the long durations of enzyme hydrolysis. Commercial enzyme cocktails Celluclast 1.5 L and Novozyme 188 were tested for hydrolysis of citrus peel and processed wood in the same assay conditions used for enzyme cocktails from crude extracts. Enzyme units of Celluclast 1.5 L and Novozyme 188 used for hydrolysis assays were equivalent to cpCelD enzyme units (based on CMC hydrolysis) present in cocktails of crude extracts. In all experiments control assays contained substrate without enzyme or enzyme without substrate. All experiments and assays were carried out in triplicate.


The inventors have used coding sequences from bacterial or fungal genomes to create chloroplast vectors. A novel PCR based method was used to clone ORFs without introns from fungal genomic DNA. E. coli expression system was used to evaluate functionality of each enzyme independently or their efficacy in enzyme cocktails before creating transgenic lines; enzymes of fungal origin were active without any need for post-translational modifications (disulfide bonds or glycosylation). The phenotypes of homoplasmic transplastomic lines were normal and produced flowers & seeds. Based on three cuttings of tobacco in one year, 49, 64 and 10,751 million units of pectate lyase and endoglucanase activity can be obtained each year in an experimental cultivar. This yield could be increased 18-fold when these enzymes are produced in commercial cultivars. Based on USDA Economic Research Service, the cost of production of Burley tobacco in 2004 was $3,981 per acre. Because most enzymes for hydrolysis of plant biomass are active at higher temperatures, it is feasible to harvest leaves and sun dry them, as reported previously for chloroplast derived xylanase25. Based on enzyme activity observed in plant crude extracts in this study, there is no need for purification. Therefore, excluding processing cost, enzymes could be produced as low as 0.008 cents for PelB, 0.006 cents for PelD per enzyme unit (as defined in the commercial source Megazyme). This is 925-1,233 fold less expensive for pectate lyase B & D, when compared with current commercial cost (Megazyme produced from C. japonicus). While this cost or yield comparison may not be the same for all chloroplast-derived enzymes, this concept provides a promising new platform for inexpensive enzyme cocktails to produce fermentable sugars from lignocellulosic biomass.


To the best of our knowledge, this is the first study using enzyme cocktails expressed in plants for hydrolysis of lignocellulosic biomass to produce fermentable sugars and direct comparison of enzyme properties produced via fermentation or in planta, using identical genes and regulatory sequences. Majority of enzyme hydrolysis studies on natural substrates like pretreated wood, corn stover or wheat straw have used commercially available enzymes3, 42 or purified recombinant enzymes spiked with purified commercial enzymes40, 43. Accurate comparison of crude extract enzyme cocktails with commercial cocktails is not possible because of their unknown enzyme compositions. Therefore, equivalent enzyme units based on CMC hydrolysis was used as a basis for general comparison. ACCELLERASE™ 1000 (Genencor) was not available for this study because of required institutional agreements. Novozyme 188 purified enzyme cocktail did not yield any detectable glucose equivalents from processed wood and 11% more glucose equivalents with citrus peel, whereas Celluclast 1.5 L yielded 10% and 137% more with both biomass substrates than the crude extract cocktail, with equivalent enzyme units based on CMC hydrolysis. It is not surprising that crude extract cocktails performed equal to or better than purified enzyme cocktails because the later are produced by submerged fermentation from selected fungal strains that secerete several enzymes, simultaneously. According to Novozymes, a careful design of a combination of single component enzymes is necessary for rational utilization of these enzyme cocktails44.


Example 9
Expression of Plant Degrading Enzymes

The teachings set forth in Examples 1-8 may be adapted for preparing expression cassettes of the heterologous gene, constructing transformation vectors; transforming chloroplasts and chloroplast expression any of a numerous list of enzymes that the inventors have identified will be helpful in degrading plant biomass sources. Also, Applicants refer to U.S. Patent Pubs 20070124830 and 20060117412 for techniques of chloroplast transformation and expression of proteins.


A non-limiting list of exemplary enzymes includes the following in Table I:









TABLE I





GOI (Genes of Interest)
















1.
Endoglucanases



a) celD (Clostridium thermocellum)



b) egI (Trichoderma reesei)



c) egl1 (Trichoderma reesei)


2.
Exoglucanase



a) celO (Clostridium thermocellum)


3.
Lipase



a) lipY (Mycobacterium tuberculosis)


4.
Pectate lyases



a) pelA (Fusarium solani)



b) pelB (Fusarium solani)



c) pelD (Fusarium solani)


5.
Cutinase



a) cut (Fusarium solani)


6.
Swollenin similar to expansins



a) swo1 (Trichoderma reesei)


7.
Xylanase



a) xyn2 (Trichoderma reesei)


8.
Acetyl xylan esterase



a) axe1 (Trichoderma reesei)


9.
Beta glucosidase



a) bgl1 (Trichoderma reesei)


10.
Mannanase



a) man1 (Trichoderma reesei)


11.
Arabinofuranosidase



a) abf1 (Trichoderma reesei)


12.
Lignin peroxidase



a) lipJ (Mycobacterium tuberculosis)









In addition to the above list, attached tables II-VII set forth a list of different enzymes that may be used in conjunction with embodiments of the invention. The EC nos. are recognized designations (http://www.chem.qmul.ac.uk/iubmb/enzyme/ and NC-IUBMB) defining each of the enzymes with cross references to relevant polypeptide sequences and encoding polynucleotide sequences. Accession Nos. pertain to identifications sequences in either Genbank (one letter followed by five digits, e.g. M12345) or the RefSeq format (two letters followed by an underscore and six digits, e.g., NT123456). Each of the sequences and related accession nos. are stored in the Corenucleotide division of the GenBank database system. An accession no. listed on table II-VII for a specific gene can be easily found by inputting the accession number in the search field of the Entrez system found at (http://www.ncbi.nlm.nih.gov/sites/gquery). In a specific embodiment, the sequences of the accession nos. specifically listed in tables II-VI include those in their original state or as revised/updated in the Genbank system as of Feb. 28, 2008. In other embodiments, it is contemplated that sequences may be revised/updated after Feb. 28, 2008 but otherwise recognized by the art as the more accurate sequence of the accession no. compared to the sequence stored prior to Feb. 28, 2008. In these other embodiments, sequences as revised but recognized as the true sequence and having at least a 95% sequence identity to the sequence as stored in database prior to Feb. 28, 2008 shall be considered as the sequence for the relevant accession no.


Applicants also incorporate by reference the ASCII text file entitled 10669-034 seqid filed with the present application. This text file contains sequence information of the accessions nos listed in Tables II-VII.


In addition, nucleotides and peptides having substantial identity to the nucleotide and amino acid sequences relating plant degrading enzymes (such as those provided in tables II-VII) used in conjunction with present invention can also be employed in preferred embodiments. Here “substantial identity” means that two sequences, when optimally aligned such as by the programs GAP or BESTFIT (peptides) using default gap weights, or as measured by computer algorithms BLASTX or BLASTP, share at least 50%, preferably 75%, and most preferably 95% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. For example, the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to effect the properties of a protein. Non-limiting examples include glutamine for asparagine or glutamic acid for aspartic acid.


The term “variant” as used herein refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits substantial identity with the nucleotide or amino acid sequence of Attachment B, preferably 75% sequence identity and most preferably 90-95% sequence identity to the sequences of the present invention: provided said variant has a biological activity as defined herein. The variant may be arrived at by modification of the native nucleotide or amino acid sequence by such modifications as insertion, substitution or deletion of one or more nucleotides or amino acids or it may be a naturally occurring variant. The term “variant” also includes homologous sequences which hybridise to the sequences of the invention under standard or preferably stringent hybridisation conditions familiar to those skilled in the art. Examples of the in situ hybridisation procedure typically used are described in (Tisdall et al., 1999); (Juengel et al., 2000). Where such a variant is desired, the nucleotide sequence of the native DNA is altered appropriately. This alteration can be made through elective synthesis of the DNA or by modification of the native DNA by, for example, site-specific or cassette mutagenesis. Preferably, where portions of cDNA or genomic DNA require sequence modifications, site-specific primer directed mutagenesis is employed, using techniques standard in the art.


In specific embodiments, a variant of a polypeptide is one having at least about 80% amino acid sequence identity with the amino acid sequence of a native sequence full length sequence of the plant degrading enzymes provided on the attached 10669-034SEDID ASCII file. Such variant polypeptides include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-terminus, as well as within one or more internal domains, of the full-length amino acid sequence. Fragments of the peptides are also contemplated. Ordinarily, a variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with a polypeptide encoded by a nucleic acid molecule shown in Attachment B or a specified fragment thereof. Ordinarily, variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, or more.


“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired identity between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).


“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by those that: (1) employ low ionic strength and high temperature for washing, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 degrees C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42 degrees C., with washes at 42 degrees C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55 degrees C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55 degrees C.


“Moderately stringent conditions” are identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50 degrees C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.


Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated Tm of the hybrid under study. The Tm of a hybrid between an polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):






T
m=81.50° C.−16.6(log10 [Na+])+0.41(% G+C)−0.63(% formamide)−600/l),


where l=the length of the hybrid in basepairs.


In a specific embodiment, stringent wash conditions include, for example, 4×SSC at 65° C., or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2×SSC at 65° C.


Example 10
Optimization of Recombinant Expressed Enzyme Cocktails

Having demonstrated by several examples that plant-degrading enzymes can be expressed in plants and bacteria, cocktails of enzymes can be produced that are especially adapted for degrading a targeted class of biomass. The inventors have discovered that certain combinations of selected enzymes are capable of hydrolyzing biomass materials in a synergistic fashion. For example, it has been discovered that two or more enzymes from a particular class work synergistically to degrade targeted material in biomass to increase the yield of fermentable sugars achieved. Based on the known composition of a target biomass sample, more than one enzyme in a particular class is included in the plant degrading cocktail. Furthermore, the amount of enzyme of a specific enzyme class known to degrade a specific compound in a biomass sample is increased relative to other enzyme in the cocktail as a function of the ratio of the specific compound/mass of biomass sample. General information concerning exemplary classes of enzymes are discussed below in relationship to the type of substrates on which they act. Equipped with the teachings and techniques discussed herein, one skilled in the art is able to determine cocktails suitable for a given biomass, and methods by which synergy of the enzymes in the cocktails can be determined.


Enzyme Assays


Most enzyme assays were determined spectrophotometrically by measuring the increase in A23530, 47, 48. Enzyme kinetics were studied to optimize substrate concentration (0-2.5 mg) under identical protein and cofactor concentrations. The reaction mixtures contained appropriate buffers, substrates and standard enzymes when commercially available. Measurements are made at different temperatures and pH. One unit of enzyme is defined as the amount of enzyme which forms 1 μmol of product per min with appropriate molar extinction coefficient. Kinetic parameters (Km & Vmax) are calculated using non linear regression using Graphpad Prism 5.0. The initial slopes of each substrate concentration are calculated where as the velocity (units/mg/min) is defined through the release of appropriate product. The temperature optimization is determined in proper buffers with required co-factors, substrates at optimal pH. In order to study the thermal stability of each enzyme, buffered enzyme samples are incubated for fixed time periods at different temperatures. Similarly, the pH optimum of each enzyme are measured at optimal temperatures using different buffers ranging from pH 6 to 10, with the same ionic strength. The stability of the crude extract of the enzyme is optimized by incubating the enzyme at the different pH. The influence of various cofactors for optimal enzyme activity is studied by conducting the reactions in the presence or absence of each cofactor, at different pH and temperature.


Endoglucanase, Cellobiohydralase and Beta-Glucosidase:


Cellulose is the most abundant renewable bioresource produced in the biosphere through photosynthetic process (˜100 billion dry tons/year)49-51. Cellulose biodegradation by cellulases and cellulosomes, produced by numerous microorganisms, represents a major carbon flow from fixed carbon sinks to atmospheric CO2 and studies have shown that the use of biobased products and bioenergy can achieve zero net carbon dioxide emission52, 53% Cellulose is a linear condensation polymer consisting of D-anhydroglucopyranose joined together by β-1,4-glycosidic bonds with a degree of polymerization (DP) from 100 to 20,000. Approximately 30 individual cellulose molecules are assembled into larger units known as elementary fibrils (protofibrils), which are packed into larger units called microfibrils, and these are in turn assembled into large cellulose fibers54, 55. The breakdown of biomass involves the release of long-chain polysaccharides, specifically cellulose and hemicellulose, and the subsequent hydrolysis of these polysaccharides into their component 5- and 6-carbon chain sugars15. One of the most important and difficult technological challenges is to overcome the recalcitrance of natural lignocellulosic materials, which must be enzymatically hydrolyzed to produce fermentable sugars2,7,8,56,57.


The mechanism of cellulose degradation involves three enzyme classes of cellulase. These include endoglucanases or 1,4-β-D-glucan-4-glucanohydrolases (EC 3.2.1.4) exoglucanase or 1,4-β-D-glucan cellobiohydrolases (E.C. 3.2.1.91), and β-glucosidase or β-glucoside glucohydrolases (E.C. 3.2.1.21)55,58,59. The combined actions of endoglucanases and exoglucanases modify the crystalline nature of cellulose surface over time, resulting in rapid changes in hydrolysis rates.60 The inventor has realized that the simultaneous action of these enzyme class on cellulose substrate completely breaks down the intramolecular β-1,4-glucosidic bonds of cellulose chains. This results in release of large amount of fermentable glucose molecules.


Endoglucanases (e.g CelD, Eg1): Endoglucanases cut at random at internal amorphous sites in the cellulose polysaccharide chain, generating oligosaccharides of various lengths and consequently expose new chain ends. Exoglucanases (e.g. CelO, Cbh2): Exoglucanases or cellobiohydrolases acts processively on the reducing or nonreducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Cellobiohydralases can also act on amorphous and microcrystalline cellulose structure having exposed cellulose chain ends. β-Glucosidases (e.g., BglA, Bgl1): It hydrolyze soluble cellobiose as well as longer cellodextrins molecules to glucose.


CelD, Egl1, EG1 (endo-glucanases) and CelO (cellobiohydrolase): Substrate: Carboxylmethylcellulose (2%) beta D-glucan (10 mg/ml), microcrystalline cellulose, Avicel, Sigma cellulose (all 50 mg/ml). At least two dilutions are made for each enzyme sample investigated in 50 mM sodium acetate buffer containing 10 mM CaCl2 and 20 μg BSA. In the assay, one dilution should release slightly more and one slightly less than 0.5 mg (absolute amount) of glucose. The sample is incubated at appropriate temperature for 30 minutes after adding 0.5 ml of substrate solution. After incubation, 0.5 ml of DNS is added to 0.5 ml of the reaction mixture followed by boiling for exactly 5 minutes in a boiling water bath along with enzyme blanks and glucose standards. Following boiling, 166 μl of 40% Rochelle salt is added and transferred immediately to a cold water bath not more than 30 minutes. The tube is mixed by inverting several times so that the solution separates from the bottom of the tube at each inversion. The mixture is measured at 575 nm in a spectrometer. The absorbance is translated (corrected if necessary by subtracting of the blank) into glucose production during the reaction using a glucose standard curve graph. Enzyme unit is defined as the amount of enzyme required to liberate 1.0 μmol per minute.32


Bgl1 (Trichoderma reesei): Beta-Glucosidase Assays:


Substrate: Cellobiose, 2-15 Mm in Sodium Acetate Buffer pH 4.8

At least two dilutions are made of each enzyme sample investigated. One dilution should release slightly more and one slightly less than 1.0 mg (absolute amount) of glucose in the reaction conditions. Add 1.0 ml of enzyme dilution in sodium acetate buffer pH 4.8 to 1.0 ml of cellobiose substrate. Incubate at 50° C. for 30-120 minutes. Stop assay by boiling in water bath for 5 minutes. Determine liberated glucose using glucose hexokinase (Sigma) method. One unit of enzyme is defined as 1.0 μmol of glucose released per minute from cellobiose. Substrate: 2-15 mM para-nitrophenyl D-glucoside in sodium acetate buffer pH 4.8. Add 0.3 ml of diluted enzyme solution to 0.6 ml 50 mM sodium acetate buffer and 0.3 ml para-nitrophenyl D-glucoside. Carry out the reaction at 50° C. for 10 minutes. Stop the reaction with 1M Na2CO3. Spectrophometrically measure the liberated p-nitrophenol at 410 nm using p-nitrophenol as standard. Construct the calibration curve for p-nitrophenol in the concentration range of 0.02-0.1 mM. Enzyme unit is defined as the amount of enzyme required to liberate 1.0 μmol of p-nitrophenol per min from pNPG.


Xylanase


Endo-xylanses: Xylan is one of the major components of the hemicellulose fraction of plant cell walls and accounts for 20-30% of their total dry mass. Unlike cellulose, xylan is a complex polymer consisting of a β-1,4-linked xylose monomers substituted with side chains. Hydrolysis of the xylan backbone is catalyzed by endob-1,4-xylanases (EC 3.2.1.8) and β-D-xylosidases (EC 3.2.1.37).6l Endoxylanases are capable of hydrolyzing the internal 1,4-β-bonds of the xylan backbone and thereby produce several xylo-oligomers of varying length. Complete hydrolysis of xylan involves endo-β-1,4-xylanase, β-xylosidase, and several accessory enzymes, such as a-L-arabinofuranosidase, α-glucuronidase, acetylxylan esterase and ferulic acid esterase.62, 63 For the biofuel industry, the inventor has realized that xylanases can be used to aid in the conversion of lignocellulose to fermentable sugars (e.g., xylose). Furthermore, hydrolysis of xylan molecules is very important step in the enzymatic hydrolysis hemicellulose and lignocellulosic materials because this gives larger accessibility for cellulases to act on exposed cellulose.


Xylanase Assay64 (Baily 1989):

Xyn2 (Trichoderma reesei)


Substrate: Oat spelt xylan (1%); Birch wood xylan (1%, boil for 5 minutes until dissolved) D-xylose (0.01 to 1 mg/ml xylose) standard graph will be prepared by using DNS method. Assay: The enzyme sample is diluted in 1% xylan suspension with a total volume reaction up to 2 ml. Then the mixture is incubated for 30-120 minutes at 50° C. After incubation, 2 ml of DNS reagent is added to the reaction mixture followed by boiling for 5 minutes. The release of xylose concentration is measured spectrophotometrically at 540 nm. The enzyme unit is calculated by using standard formula64.


Cutinase:


Cutinase from the phytopathogenic fungus Fusarium solani pisi is an example of a small carboxylic ester hydrolase that bridges functional properties between lipases and esterases. Cutin, a polyester composed of hydroxy and hydroxy epoxy fatty acids containing 16 and 18 carbon atoms, is the major structural component of the protective barrier covering the surface of the aerial parts of plants65, 66. Cutinases not only degrade cutin polymers but also a large variety of short and long chain triacylglycerols are rapidly hydrolyzed. The enzyme belongs to the family of serine hydrolases containing the so called α/β hydrolase fold.67, 68 Hydrolyses of cutin, lipase and triacylglycerols molecules that present in large amount in citrus peel waste gives tremendous accessibility for enzymes like pectinases, cellulases and xylanases. Therefore cutinase is important enzyme component in the enzyme hydrolyses of citrus peel for biofuel production.


Cutinase assay67, 69: Substrate: p-nitrophenyl butyrate and p-nitrophenyl palmitate (0.01% or 10 mM). The enzyme is extracted from transplastomic plants in 100 mM Tris-HCl buffer (pH7.0) and 0.03% Triton X-100 will be added at the time of initiating enzyme assay. Reactions are performed by incubating for 10-15 min at 30° C. in a tube containing 1 ml of substrate (100 mM Tris-HCl, pH 7, 0.03% Triton X-100, and 0.01% p-nitrophenol butyrate and various amount of enzyme sample (ice cold). Release of p-nitrophenol is measured at 405 nm using p-nitrophenol as standard. Background activity is subtracted from the absorption reading if necessary. One unit of enzyme activity was defined as the amount that degrades 1 μmol of substrate per minute under standard conditions.


Mannanase:


Mannanase is used in the paper and pulp industry, for the enzymatic bleaching of softwood pulp, in the detergent industry as a stain removal booster, in the coffee industry for hydrolysis of coffee mannan to reduce the viscosity of coffee extracts, in oil drilling industry to enhance the flow of oil or gas, in oil extraction from coconut meat, in the textile industry for processing cellulosic fibers and in the poultry industry to improve the nutritional value of poultry feeds.70


Cell-wall polysaccharides can be converted into fermentable sugars through enzymatic hydrolysis using enzymes such as cellulases and hemicellulases and the fermentable sugars thus obtained can be used to produce lignocellulosic ethanol. Mannanase is a hemicellulase. Hemicellulose is a complex group of heterogeneous polymers and represents one of the major sources of renewable organic matter. Mannans are one of the major constituent groups of hemicellulose and are widely distributed in hardwoods and softwoods, seeds of leguminous plants and in beans. Hemicelluloses make up 25-30% of total wood dry weight. Hemicelluloses in softwoods are mainly galactoglucomannan, containing mannose/glucose/galactose residues in a ratio of 3:1:1 and glucomannan with mannose/glucose residues in the ratio of 3:171. Mannanases are endohydrolases that cleave randomly within the 1,4-β-D mannan main chain of galactomannan, glucomannan, galactoglucomannan, and mannan. Mannanases hydrolyzes the β-D-1,4 mannopyranoside linkages in β-1, 4 mannans. The main products obtained during the hydrolysis of mannans by β-mannanases are mannobiose and mannotriose. Additional enzymes, such as β-glucosidases and α-galactosidases are required to remove side chain sugars that are attached at various points on mannans. A vast variety of bacteria, actinomycetes, yeasts and fungi are known to produce mannanase. Among bacteria, mostly gram-positive, including various Bacillus species and Clostridia species and few strains of gram negative bacteria, viz. Vibrio and Bacteroides have also been reported. The most prominent mannan degrading group among fungi belongs to genera Aspergillus, Agaricus, Trichoderma and Sclerotium.70


The assay procedure for determination of the activity of Mannanase involves the incubation of the crude enzyme extract with the substrate (Galactomannan from Locust bean gum as a substrate). After the enzyme reaction the reducing sugars liberated are quantified and the enzyme activity is measured. Locust bean gum (0.5%) will be dissolved in citrate buffer (pH 5.3) and heated until boiled; this mixture is used as the substrate. The crude enzyme extract is incubated with the substrate at 50° C. for 5 minutes. The reducing sugars liberated in the enzyme reaction is assayed by adding Dinitro salicylic acid-reagent boiling for 5 min, cooling and measuring the absorbance at 540 nm72.


Another assay method involves carob galactomannan (0.2%) as substrate in sodium acetate buffer (pH 5). Crude enzyme extract is incubated with the substrate at 40° C. for 10 minutes. The reducing sugars liberated are measured by Nelson-Somogyi method and the enzyme activity is quantified73. In gel diffusion assay, gel plates are prepared by dissolving 0.05% (w/v) locust bean gum in citrate phosphate buffer (pH 5.0) along with Phytagar. Crude enzyme extract is transferred to the gel plates and incubated for 24 hours. Gels will be stained by using Congo red. Cleared zones (halos) on the plates indicated endo-beta-mannanase activity74.


Arabinofuranosidase 1 (ABF1)


ABF1 has been shown to have numerous potential uses. L-arabinose is found throughout many different plant tissues in small amounts but strategically placed as side groups. ABF1 facilitates the breakdown of these side chains and cross-linking within the cell wall to work synergistically with other enzymes such as hemicellulases. This can increase the availability of fermentable sugars for biofuel production from biomass or pulp and paper production. This enzyme can increase the digestibility of livestock feed, has been used in the clarification of fruit juices and can aid in the aromatization of wines. Finally, ABF1 has possibilities as a food additive for diabetics due to the sweet taste and inhibitory effect on the digestion of sucrose75.


ABF1 was isolated from the fungus T. reesei and expressed in Escherichia coli in the pLD plasmid. The activity of this enzyme is assayed through the use of p-nitrophenyl-α-L-arabinofuranoside substrate in a 0.05M citrate buffer incubated at 50° C. for 10 minutes with enzyme crude extracts from plants or E. coli. The reaction is stopped with 1M Na2CO3 and the resultant p-nitrophenol is measured by spectrophotometer at 400 nm and compared to the positive standard (either p-nitrophenol or ABF, both of which are available commercially) to determine quantity. The pH and temperature optima studies are performed with the same substrate and measured in the same fashion 76. The activity of this enzyme has been known to be inhibited certain metal ions such as Cu2+, Hg2+, detergents and many chelating and reducing agents.75


Lignin Peroxidase (LipJ)


The enzyme Lignin peroxidase, commonly known as LipJ, remains novel and desirable for advancing the production of biofuel enzymes by means of transplastomic tobacco. Expression of LipJ should expand the spectra of exploitable biomass sources by hydrolysis of the inedible lignin and cellulose rich portions of biomass such as corn, sugarcane and wheat. Sources previously occluded as sources for biofuel due their high lignin content e.g. waste lumber, wood chips, peels from commercially prepared fruit and vegetables, could be hydrolyzed with LipJ. LipJ also advances biofuels production by ablating the intricate lignin structures within plant materials from inhibiting access to more valuable biomass substrates that provide valuable commercial, chemical and pharmaceutical products. These products are as of yet, limited in yield because of the protective nature of lignin surrounding them within biomass sources.


LipJ, was isolated from genomic DNA of Mycobacterium tuberculosis, expressed in a plasmid functional in both chloroplasts and E. coli. Three cultivars of transplastomic tobacco are expressing this vector in the primary selection round and await confirmation of chloroplast expression. Protein assays in E. coli have been designed for qualitative & quantitative analysis of LipJ expression, which is optimized for chloroplast derived LipJ.


Lipase (Lip Y)

Lipase Y (LipY), from Mycobacterium Tuberculosis, is a water soluble enzyme that catalyzes the hydrolysis of ester bonds and long-chain triacylglycerols, making it an ideal candidate for biofuel production. LipY is a membrane protein for Mycobacterium Tuberculosis and studies have already shown a humoral response will occur when LipY is combined with the serum of tuberculosis patients; therefore, LipY has the added benefit of being a potential vaccine for tuberculosis 77.


One simple assay for determining the activity of the cloned gene is a plate assay involving the fluorescent dye rhodamine B and a crude extract of lyses cells. If the gene was properly integrated and the protein folded correctly the assay will display an orange color when illuminated under a specific wavelength of light 80. Another assay measures the release of p-nitrophenol when p-nitrophenylstearate is incubated in various concentrations of E. coli or plant cell extract81.


Example 11
Expression of Plant Degrading Enzymes in Commercial Cultivars

Tobacco chloroplasts were transformed by microprojectile bombardment (biolistic transformation) as described before. Transgene integration was confirmed by PCR analysis using primers used in previous studies. Southern blot analysis was conducted to confirm homoplasmy. Tobacco (Nicotiana tabacum var. Petit Havana/LAMD/TN90) seeds were aseptically germinated on MSO medium in Petri dishes. Germinated seedlings were transferred to magenta boxes containing MSO medium. Leaves at 3-7 leaf stage of plant growth were cut and placed abaxial side up on a Whatman filter paper laying on RMOP medium in Petri plates (100×15 mm) for bombardment. Gold microprojectiles (0.6 μm) were coated with plasmid DNA (tobacco chloroplast expression vector) and biolistic mediated transformation were carried out with the biolistic device PDS1000/He (Bio-Rad) as published. After bombardment, leaves were incubated in dark for 48 hours to recover from damage. After 48 hours in dark, the bombarded leaves of Petit Havana (experimental cultivar) or LAMD and TN90 (commercial cultivar) were cut into 5 mm pieces and placed on plates (bombarded side in contact with medium) containing RMOP with 500, 150 and 200 mg/l of spectinomycin respectively for the first round of selection. After 4-5 weeks, resistant shoots will appear, whereas untransformed cells will die. Resistant shoots will be transferred to new RMOP-Spectinomycin plates and subjected to subsequent rounds of selection. The putative transplastomic shoots were confirmed by PCR and Southern analysis. Expression of cell wall degrading enzymes were confirmed by their respective assays and for those that antibody was available, Western blot analysis was performed.


The commercial cultivars yielded 40 metric tons biomass of fresh leaves as opposed to 2.2 tons in experimental cultivar Petit Havana. The commercial cultivars yield biomass 18 fold more than the experimental cultivar (Cramer et al., 1999). LAMD-609 is a low nicotine hybrid produced by backcrossing a Maryland type variety, MD-609, to a low nicotine-producing burley variety, LA Burley 21 (Collins et al., 1974), Tennessee 90 (TN 90) is a commercial cultivar used by Philip Morris (Lancaster Laboratories, PA). Both experimental cultivars LAMD and TN 90 were transformed with genes encoding biomass degrading enzymes. Although it is more challenging to transform these cultivars than the experimental cultivar, we succeeded in transforming both commercial cultivars with a number of genes encoding biomass degrading enzymes pectate lyases, cellulases, xylanases and endoglucanases. Transplastomic lines of commercial cultivars were homoplasmic, fertile and activities of expressed enzymes were similar to the experimental cultivar except that their biomass yield was much higher than Petit Havana. Table 8 shows enzymes that have been successfully introduced in plants, expressed and assayed for activity in experimental and commercial cultivars.


PCR was done using DNA isolated from leaf material of control and putative transgenic plants in order to distinguish true chloroplast transformants from nuclear transformants or mutants. Two separate PCR reactions was set up, one reaction checked for the integration of selectable marker gene into the chloroplast genome and the second checked integration of the transgene expression cassette. In order to test chloroplast integration of the transgenes, one primer (3M) will land on the aadA gene while another (3P) will land on the native chloroplast genome. No PCR product was obtained with nuclear transgenic plants or mutants using this set of primers. This screening is important for eliminating mutants and nuclear transformants. In order to conduct PCR analysis in transgenic plants, total DNA from unbombarded and transgenic plants was isolated as described by DNeasy plant mini kit (Qiagen). Integration of transgene expression cassette was tested using 5P/2M primer pair. Primer 5P lands in the aadA gene and 2M in the trnA, therefore, the PCR product showed whether the gene of interest had been introduced into the chloroplast genome via the homologous recombination process. A similar strategy has been used successfully by PI lab to confirm chloroplast integration of several foreign genes. The leaf pieces from PCR-positive shoots was further selected for a second round in order to achieve homoplasmy.


Southern blots were done to test homoplasmy. There are several thousand copies of the chloroplast genome present in each plant cell. When foreign genes are inserted into the chloroplast genome, not all chloroplasts will integrate foreign DNA resulting in heteroplasmy. To ensure that only the transformed genome exists in transgenic plants (homoplasmy), the selection process was continued. In order to confirm homoplasmy at the end of the selection cycle, total DNA from transgenic plants was probed with the radiolabeled chloroplast flanking sequences (the trnI-trnA fragment) used for homologous recombination. If wild type genomes are present (heteroplasmy), the native fragment size was observed along with transformed genomes. Presence of a large fragment due to the insertion of foreign genes within the flanking sequences and the absence of the native small fragment should confirm homoplasmy.



FIG. 6 shows the generation of transplastomic tobacco commercial cultivars (A) Rooting of CelD LAMD shoot (B) CelD LAMD transplastomic plants growing in the green house (C) CelD LAMD transplastomic plants showing normal flowering (D) CelD TN90 primary transformant (E) Second round of regeneration for CelD TN90 (F) Rooting of PelB TN90 (G-I) First, second and third round of regeneration for PelB LAMD (J) Rooting of PelD LAMD shoot (K) PelD LAMD transplastomic plants growing in green house (L) PelD LAMD transplastomic plant showing normal flowering (M) Rooting of eg1LAMD shoot (N) eg1 LAMD transplastomic plant growing in pots.



FIG. 7 shows confirmation of homoplasmy by southern blots using tobacco flanking probe (A) CelD LAMD (B) PelB LAMD (C) PelB TN90 (D) PelD LAMD and (E) eg1 LAMD (UT: Untransformed plant; Numbers: Transplastomic lines and B: Blank). FIG. 8 shows enzymatic activity of pectate lyase B and D in Petit Havana, TN90 and LAMD tobacco cultivars (A) PelB (B) PelD Note: The leaf material used for the analysis of enzyme activity for TN90 and LAMD tobacco cultivars were harvested from in vitro plants, whereas the leaf material for Petit Havana is from the green house. Since the transgene is controlled by psbA, with light and developmental regulatory elements, expression levels in commercial cultivars are expected to be higher when transferred to the green house.









TABLE 8







Summary of Successful Transformation, Expression and Active Recombinantly


Expressed Enzymes





















Assay










with
Assay







Frozen

E. coli

with


Gene
PCR
Southern
At what
No. of
material
extract
plant
Plant


Name
Positive
Status
stage
plants
(gms)
protocols
extract
Health





celD
Yes, Petit
Homoplasmy
Collected
T0 seeds
400
Yes
Yes
Normal



Havana

seeds
germinated



(PH)



Yes
Homoplasmy
Collected
T0 seeds
250

Yes
Normal



(LAMD)

seeds
germinated



Yes (TN90)
Not Checked
2nd Round
4 clones


Yes
Normal





of selection


pelB
Yes (PH)
Homoplasmy
Collected
T0 seeds
700
Yes
Yes
Normal





seeds
germinated



Yes
Homoplasmy
Rooting
18 plants


Yes
Normal



(LAMD)



Yes (TN90)
Homoplasmy
Ready for
30 plants


Yes
Normal





Green





house





production


pelD
Yes (PH)
Homoplasmy
Collected
T0 seeds
600
Yes
Yes
Normal





seeds
germinated



Yes
Homoplasmy
Collected
T0 seeds
340

Yes
Normal



(LAMD)

seeds
germinated


pelA
Yes (PH)
Homoplasmy
Ready for
 9

Yes
Yes
Normal





Green





house





production


egI
Yes (PH)
Homoplasmy
Ready for
 9

Yes
Yes
Normal





Green





house





production



Yes
Homoplasmy
Ready for
 3


Yes
Normal



(LAMD)

Green





house





production


Egl1
Yes (PH)
in progress
Rooting
20

in
in
Normal



Yes (TN90)
in progress
2nd round
3 clones

progress
progress
Normal





of selection


Xyn2
Yes (PH)
Homoplasmy
Ready for
 7

Yes
Yes
Normal





Green





house





production


Swo1
Yes (PH)
Homoplasmy
Rooting
20

Yes
Yes
Thin










leaves


Bgl1
Yes (PH)
Homoplasmy
Ready for
20

Yes
in
Normal





Green



progress





house





production


Cutinase
Yes (PH)
in progress
Rooting
15

Yes
in
Leaves









progress
are thin


cello
Yes (PH)
Heteroplasmy
Rooting
20

Yes
in
Normal









progress



Screening
in progress
2nd round
5 clones

Yes
in
Normal



(LAMD)

of selection



progress


Axe1
Bombarded

In RMOP


Yes



in all

selection



cultivars

medium


lip Y
Bombarded

In RMOP


Yes



in all

selection



cultivars

medium


lipJ
Bombarded

In RMOP


yes



in all

selection



cultivars

medium


Man1
Bombarded

In RMOP


Yes



in all

selection



cultivars

medium


Abf1
Bombarded

In RMOP



in all

selection



cultivars

medium









The disclosures of the cited patent documents, publications and references, including those referenced in Tables II-VII, are incorporated herein in their entirety to the extent not inconsistent with the teachings herein. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.


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  • 23. Svab, Z. & Maliga, P. Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proceedings of the National Academy of Sciences 104, 7003-7008 (2007).

  • 24. Gray, B. N., Ahner, B. A., & Hanson, M. R. High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions. Biotechnology and Bioengineering xxx, xxx (2008).

  • 25. Leelavathi, S., Gupta, N., Maiti, S., Ghosh, A., & Siva Reddy, V. Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme. Molecular Breeding 11, 59-67 (2003).

  • 26. Yu, L. X. et al. Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco. Journal of Biotechnology 131, 362-369 (2007).

  • 27. Brixey, P. J., Guda, C., & Daniell, H. The chloroplast psbA promoter is more efficient in Escherichia coli than the T7 promoter for hyperexpression of a foreign protein. Biotechnology Letters 19, 395-400 (1997).

  • 28. Daniell, H., Ruiz, O. N., & Dhingra, A. Chloroplast Genetic Engineering to Improve Agronomic Traits. Methods Mol Biol 286, 111-138 (2005).

  • 29. Verma, D., Samson, N. P., Koya, V., & Daniell, H. A protocol for expression of foreign genes in chloroplasts. Nat. Protocols 3, 739-758 (2008).

  • 30. Guo, W., Gonzalez-Candelas, L., & Kolattukudy, P. E. Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f. sp. pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris. J. Bacteriol. 177, 7070-7077 (1995).

  • 31. Miller, G. L. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry 31, 426-428 (1959).

  • 32. Ghose, T. K. Measurement of cellulase activities. Pure and Applied Chemistry 59, 257-268 (1987).

  • 33. Arlen P A et al. Field production and functional evaluation of chloroplast-derived interferon-alpha2b. Plant Biotech J 5, 511-525 (2007).

  • 33. Cramer, C. L., Boothe, J. G. & Oishi, K. K. Transgenic plants for therapeutic proteins: linking upstream and downstream strategies. Curr. Top. Microbiol. Immunol. 240, 95-118 (1999).

  • 34. Ruhlman, T. F., Ahangari, R. F., Devine, A. F., Samsam, M. F., & Daniell, H. Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts—oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotech J 5, 495-510 (2007).

  • 35. Bally, J. et al. Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins. Plant Biotech J 6, 46-61 (2008).

  • 36. Chauvaux, S. et al. Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. Biochem. J. 265, 261-265 (1990).

  • 37. Zverlov, V. V., Velikodvorskaya, G. A., & Schwarz, W. H. A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose. Microbiology 148, 247-255 (2002).

  • 38. Irwin D C, Spezio, M., Walker L P, & Wilson, D. B. Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects. Biotechnology and Bioengineering 42, 1002-1013 (1993).

  • 39. Zhou, S. & Ingram, L. O, Synergistic Hydrolysis of Carboxymethyl Cellulose and Acid-Swollen Cellulose by Two Endoglucanases (CelZ and CelY) from Erwinia chrysanthemi. J. Bacteriol. 182, 5676-5682 (2000).

  • 40. Gusakov A V et al. Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose. Biotechnology and Bioengineering 97, 1028-1038 (2007).

  • 41. Yapo, B. M., Lerouge, P., Thibault, J. F., & Ralet, M. C. Pectins from citrus peel cell walls contain homogalacturonans homogenous with respect to molar mass, rhamnogalacturonan I and rhamnogalacturonan II. Carbohydrate Polymers 69, 426-435 (2007).

  • 42. Rosgaard, L., Pedersen S, & Meyer, A. S. Comparison of different pretreatment strategies for enzymatic hydrolysis of wheat and barley straw. Appl. Biochem. Biotechnol. 143, 284-296 (2007).

  • 43. Selig, M. J., Knoshaug, E. P., Adney, W. S., Himmel, M. E., & Decker, S. R. Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities. Bioresource Technology 99, 4997-5005 (2008).

  • 44. Rosgaard, L. et al. Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates. Biotechnol. Prog 23, 1270-1276 (2007).

  • 45. Guo, W., Gonzalez-Candelas, L., & Kolattukudy, P. E. Identification of a NovelpelDGene Expressed Uniquely in Planta by Fusarium solanif. sp. pisi (Nectria haematococca, Mating Type VI) and Characterization of Its Protein Product as an Endo-Pectate Lyase. Archives of Biochemistry and Biophysics 332, 305-312 (1996).

  • 46. Soliday, C. L., Flurkey, W. H., Okita, T. W., & Kolattukudy, P. E. Cloning and structure determination of cDNA for cutinase, an enzyme involved in fungal penetration of plants. Proceedings of the National Academy of Sciences of the United States of America 81, 3939-3943 (1984).

  • 47. Daniell, H., Datta, R., Varma, S., Gray, S., & Lee, S. B. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat Biotech 16, 345-348 (1998).

  • 48. Kumar, S. & Daniell, H. Engineering the Chloroplast Genome for Hyperexpression of Human Therapeutic Proteins and Vaccine Antigens in Recombinant Gene Expression 365-383 2004).

  • 49. Crawford, M. S. & Kolattukudy, P. E. Pectate lyase from Fusarium solani f. sp. pisi: Purification, characterization, in vitro translation of the mRNA, and involvement in pathogenicity. Archives of Biochemistry and Biophysics 258, 196-205 (1987).

  • 50. Gonzalez-Candelas, L. & Kolattukudy, P. E. Isolation and analysis of a novel inducible pectate lyase gene from the phytopathogenic fungus Fusarium solani f. sp. pisi (Nectria haematococca, mating population VI). J. Bacteriol. 174, 6343-6349 (1992).



REFERENCE LIST FOR EXAMPLES 10-11
Reference List



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  • 32. Ghose, T. K. Measurement of cellulase activities. Pure and Applied Chemistry 59, 257-268 (1987).

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  • 38. Chauvaux, S. et al. Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. Biochem. J. 265, 261-265 (1990).

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TABLE II





No.
Source
Gene
Accession No.


















1

Aspergillus niger CBS

1,4-beta-D-arabinoxylan
XM_001389961



513.88
arabinofuranohydrolase axhA


2

Aspergillus niger CBS

endo-1,4-beta-xylanase A
XM_001389959



513.88
precursor (xynA)


3

Aspergillus niger

xylanase B
DQ174549


4

Aspergillus niger

xylanase (XYNB)
AY536639


5

Aspergillus niger

xylanase (XYN6)
AY536638


6

Aspergillus niger

xylanase (XYN4)
U39785


7

Aspergillus niger

xylanase (XYN5)
U39784


8

Aspergillus fumigatus

XynC
DQ156555


9

Aspergillus fumigatus

XynB
DQ156553


10

Bacillus licheniformis

I5 beta-1,4-endoxylanase
DQ520129




(xyn11)


11

Cryptococcus flavus

endo-1,4-beta xylanase
EU330207



isolate I-11
(XYN1)


12

Trichoderma viride

endo-1,4-beta-xylanase (xyn2)
EF079061



strain AS 3.3711


13

Thermoascus aurantiacus

xynA
AJ132635


14

Agaricus bisporus

xlnA
Z83310


15

Thermobifida alba

xylA
Z81013


16

Bacillus subtilis

eglS gene for endo-1,4-beta-
Z29076




glucanase


17

Chaetomium cupreum

endo-1,4-beta-xylanase
EF026978


18

Paenibacillus polymyxa

xyn D and glu B genes for
X57094




endo-beta-(1,4)-xylanase and




endo-beta-(1,3)(1,4)-glucanase


19

Neocallimastix frontalis

xylanase
DQ517887



strain k13


20

Penicillium citrinum

xynA gene for endo-1,4-beta-
AB198065




xylanase


21

Agaricus bisporus

endo-1,4-beta xylanase
Z83199


22

Bacillus sp. (137)

endo-beta-1,4-xylanase
Z35497


23

Bacillus pumilus

xynA
X00660


24

Aeromonas punctata

XynX
AB015980


25

Penicillium canescens

endo-1,4-beta-xylanase gene
AY756109


26

Cochliobolus carbonum

endo-beta-1,4 xylanase
AY622513




(XYL4)


27

Aspergillus cf. niger

endo-1,4-beta-xylanase B
AY551187



BCC14405
(xylB)


28

Bacillus alcalophilus

beta-1,4-xylanase (xynT)
AY423561



strain AX2000


29

Trichoderma viride

endo-1,4-beta-xylanase
AY370020



strain YNUCC0183
(XYL1)


30

Thermotoga maritima

endo-1,4-beta-xylanase B
AY339848


31

Trichoderma viride

endo-1,4-beta-xylanase
AY320048



strain YNUCC0183


32

Gibberella zeae

endo-1,4-beta-xylanase (xylA)
AY289919


33

Aeromonas caviae

xynA gene for xylanase I
D32065


34

Bacillus pumilus strain

beta-1,4-xylanase (xynK) gene,
AF466829



TX703


35

Fusarium oxysporum f.

xylanase 4 protein (xyl4)
AF246831



sp. lycopersici


36

Fusarium oxysporum f.

xylanase 5 protein (xyl5) gene
AF246830



sp. lycopersici


37

Penicillium

endo-1,4-beta-D-xylanase A
AF249328




purpurogenum

(XynA)


38

Streptomyces sp. S38

xyl1 gene for endo-1,4-beta-
X98518




xylanase


39

Bacillus sp. NBL420

endo-xylanase (xylS)
AF441773


40

Bacillus

endo-beta-1,4-xylanase (xynA)
U15985




stearothermophilus



41

Phanerochaete

endo-1,4-B-xylanase B (xynB)
AF301902 to




chrysosporium strain


AF301905



ME446


42

Thermoascus aurantiacus

endo-1,4-beta-xylanase A
AF127529




precursor (xynA) gene


43

Neocallimastix

endo-1,4-beta-xylanase (xynC)
AF123252




patriciarum

gene


44

Streptomyces avermitilis

endo-1,4-beta-xylanase (xyl30)
AF121865




gene


45

Cochliobolus carbonum

beta-1,4-xylanase precursor
U58916




(XYL3) gene


46

Trichoderma reesei

beta-xylanase (XYN2)
U24191


47

Aspergillus tubingensis

xylanase (xlnA) gene
L26988


48

Thermomonospora fusca

endo 1,4-beta-D xylanase gene
U01242



YX


49

Aspergillus fumigatus

xylosidase:
XM_750558



Af293
arabinofuranosidase


50

Aspergillus fumigatus

beta-xylosidase XylA
XM_747967



Af293


51

Aspergillus fumigatus

beta-xylosidase
XM_744780



Af293


52

Aspergillus fumigatus

Xld
DQ156554


53

Vibrio sp. XY-214

xloA
AB300564


54

Aspergillus niger

xlnD
Z84377


55

Bacteroides ovatus

xylosidase/arabinosidase gene
U04957


56

Aspergillus clavatus

xylosidase:
XM_001275592



NRRL 1
arabinofuranosidase


57

Aspergillus clavatus

beta-xylosidase
XM_001268537



NRRL 1


58

Neosartorya fischeri

beta-xylosidase
XM_001261557



NRRL 181


59

Aspergillus niger CBS

xylosidase xlnD
XM_001389379



513.88


60

Aspergillus oryzae

beta-xylosidase A
AB013851


61

Aspergillus oryzae

beta-1,4-xylosidase
AB009972


62

Vibrio sp. XY-214

xloA
AB300564


63

Thermoanaerobacterium

xylosidase
EF193646



sp. ‘JW/SL YS485’


64

Penicillium herquei

xylosidase
AB093564


65

Bifidobacterium

beta-xylosidase (bxyL) gene
DQ327717




adolescentis strain Int57



66

Aspergillus awamori

beta-xylosidase
AB154359


67

Bacillus pumilus IPO

xynB gene for beta-xylosidase
X05793


68

Pyrus pyrifolia PpARF2

alpha-L-arabinofuranosidase/
AB195230




beta-D-xylosidase


69

Clostridium stercorarium

bxlA gene for beta-xylosidase A
AJ508404


70

Clostridium stercorarium

bxlB gene for beta-xylosidase B
AJ508405


71

Aeromonas punctata

xysB, xyg genes for xylosidase
AB022788




B, alpha-glucuronidase,


72

Clostridium stercorarium

xynA gene for endo-xylanase
AJ508403


73

Clostridium stercorarium

xyl43B gene for beta-
AB106866




xylosidase


74

Talaromyces emersonii

beta-xylosidase (bxl1) gene
A439746


75

Thermoanaerobacterium

beta-xylosidase (xylB) and
AF001926



sp. ‘JW/SL YS485’
xylan esterase 1 (axe1) genes


76

Streptomyces

beta-xylosidase
AB110645




thermoviolaceus



77

Selenomonas

xylosidase/arabinosidase (Xsa)
AF040720




ruminantium

gene


78

Bacillus pumilus

xylan 1,4-beta-xylosidase
AF107211




(xynB)


79

Bacillus

b-xylosidase and gene for
D28121




stearothermophilus

xylanase


80

Azospirillum irakense

xylosidase/arabinofuranosidase
AF143228




(xynA) gene


81

Cochliobolus carbonum

major extracellular beta-
AF095243




xylosidase (XYP1) gene


82

Streptomyces lividans

BxlS (bxlS), BxlR (bxlR),
AF043654




BxlE (bxlE), BxlF (bxlF),




BxlG (bxlG), and BxlA (bxlA)




genes


83

Bacillus sp. KK-1

beta-xylosidase (xylB) gene
AF045479


84

Aspergllus nidulans

xlnD
Y13568


85

T. reesei

beta-xylosidase
Z69257


86

T. reesei

alpha-L-arabinofuranosidase
Z69252


87

Clostridium stercorarium

xylA gene encoding xylosidase
D13268


88

Butyrivibrio fibrisolvens

beta-D-xylosidase/alpha-L-
M55537




arabinofuranosidase gene


89

Thermoanaerobacter

beta-xylosidase (xynB)
M97883


90

Paenibacillus sp. W-61

exo-oligoxylanase
AB274730


91

Aspergillus niger CBS

arabinofuranosidase B abfB
XM_001396732



513.88


92

Aspergillus niger CBS

1,4-beta-D-arabinoxylan
XM_001389961



513.88
arabinofuranohydrolase axhA


93

Aspergillus niger

alpha-L-arabinofuranosidase
U39942




(ABF2)


94

Aspergillus niger

alpha-L-arabinofuranosidase
L29005




(abfA)


95
Synthetic contruct
Alpha-L-arabinofuranosidase
BD143577




gene


96
Synthetic contruct
Alpha-L-arabinofuranosidase
BD143576




gene


97

Aureobasidium pullulans

alpha arabinofuranosidase
AY495375




(abfA) gene


98

Geobacillus

alpha-L-arabinofuranosidase
EF052863




stearothermophilus strain

(abf) gene



KCTC 3012


99

Hypocrea jecorina strain

Abf2
AY281369



QM6a


100

Aspergillus sojae

alpha-L-arabinofuranosidase
AB033289


101
Uncultured bacterium
arabinofuranosidase (deAFc)
DQ284779



clone LCC-1
gene


102

Penicillium

alpha-L-arabinofuranosidase 2
EF490448




purpurogenum

(abf2) gene


103

Acremonium

Novel Alpha-L-
DD354226 to




cellulolyticus

arabinofuranosidase
DD354230


104

Fusarium oxysporum f.

abfB gene for alpha-L-
AJ310126



sp. dianthi
arabinofuranosidase B


105

Clostridium stercorarium

arfA gene for alpha-
AJ508406




arabinofuranosidase


106

Streptomyces

stxIV, stxI genes for alpha-L-
AB110643




thermoviolaceus

arabinofuranosidase, xylanase I


107

Bifidobacterium longum

arabinofuranosidase (abfB)
AY259087



B667
gene


108

Aspergillus oryzae

Alpha-L-arabinofuranosidase
BD143578




gene


109

Aspergillus oryzae

Alpha-L-arabinofuranosidase
BD143575




gene


110

Clostridium

alpha-L-arabinofuranosidase
AY128945




cellulovorans

ArfA, beta-galactosidase/alpha-




L-arabinopyranosidase BgaA


111

Bacillus

alpha-L-arabinofuranosidase
AF159625




stearothermophilus

(abfA) gene


112

Aspergillus oryzae

abfB for alpha-L-
AB073861



strain: RIB40
arabinofuranosidase B


113

Aspergillus oryzae

abfB for alpha-L-
AB073860



strain: HL15
arabinofuranosidase B


114

Penicillium

alpha-L-arabinofuranosidase
AF367026




purpurogenum

(abf) gene


115

Cochliobolus carbonum

alpha-L-arabinofuranosidase
AF306764




(ARF2) gene


116

Cochliobolus carbonum

alpha-L-arabinofuranosidase
AF306763




(ARF1) gene


117

Cytophaga xylanolytica

alpha-L-arabinofuranosidase
AF028019



XM3
ArfII (arfII) gene


118

Cytophaga xylanolytica

alpha-L-arabinofuranosidase
AF028018



XM3
ArfI (arfI) gene


119

Clostridium stercorarium

alpha-L-arabinofuranosidase
AF002664




(arfB) gene


120

Aspergillus niger

tannase (TanAni)
DQ185610


121

Neosartorya fischeri

tannase and feruloyl esterase
XM_001262461



NRRL 181
family protein (NFIA_029970)


122

Neosartorya fischeri

tannase and feruloyl esterase
XM_001261621



NRRL 181
family protein (NFIA_027990)


123

Neosartorya fischeri

tannase and feruloyl esterase
XM_001257320



NRRL 181
family protein (NFIA_047590)


124

Talaromyces stipitatus

faeC gene for ferulic acid
AJ505939




esterase


125

Aspergillus niger

faeB gene for feruloyl esterase
AJ309807


126

Aspergillus awamori

AwfaeA gene for
AB032760




feruloylesterase


127

Penicillium

fae-1
AB206474




chrysogenum



128

Neurospora crassa

ferulic acid esterase, type B
AJ293029


129

Volvariella volvacea

acetyl xylan esterase
DQ888226


130

Aspergillus fumigatus

acetyl xylan esterase
XM_750185



Af293


131

Aspergillus niger CBS

acetyl xylan esterase axeA
XM_001395535



513.88


132

Neosartorya fischeri

acetyl xylan esterase (Axe1)
XM_001258648



NRRL 181


133

A. niger

acetyl xylan esterase (axe A)
A22880


134

Didymella rabiei

cut gene for cutinase
X65628


135

Penicillium

acetyl xylan esterase (axeI)
AF529173




purpurogenum



136

Aspergillus oryzae

AoaxeA gene for acetyl xylan
AB167976





esterase



137

Fibrobacter

acetyl xylan esterase Axe6A
AF180369




succinogenes subsp.

(axe6A) gene




succinogenes S85



138

Aspergillus ficuum

acetyl xylan esterase gene
AF331757


139

Bacillus pumilus

axe gene for acetyl xylan
AJ249957




esterase


140

Trichoderma reesei

acetyl xylan esterase
Z69256


141

Thermoanaerobacterium

beta-xylosidase (xylB) and
AF001926



sp. ‘JW/SL YS485’
xylan esterase 1 (axe1) genes


142

Streptomyces

stxII, stxIII genes for xylanase
AB110644




thermoviolaceus

II, acetyl xylan esterase


143

Streptomyces

stxIV, stxI genes for alpha-L-
AB110643




thermoviolaceus

arabinofuranosidase, xylanase I


144

Streptomyces lividans

acetyl-xylan esterase (axeA)
M64552




and xylanase B (xlnB) genes


145
Abiotrophia para-
XynC gene for acetyl esterase
AB091396



adiacens


146

Orpinomyces sp. PC-2

acetyl xylan esterase A (AxeA)
AF001178


147

Caldocellum

xylanase A (XynA), beta-
M34459




saccharolyticum

xylosidase (XynB) and acetyl




esterase (XynC) genes


148

Talaromyces emersonii

alpha-glucuronidase (aGlu)
AF439788




gene


149

Aspergillus niger

aguA gene for alpha-
AJ290451




glucuronidase


150

Aspergillus fumigatus

alpha-glucuronidase
XM_748126



Af293


151

Aspergillus niger CBS

alpha-glucuronidase aguA
XM_001401166



513.88


152

Aspergillus clavatus

alpha-glucuronidase
XM_001274705



NRRL 1


153

Neosartorya fischeri

alpha-glucuronidase
XM_001259233



NRRL 181


154

Neosartorya fischeri

beta-galactosidase
XM_001259223



NRRL 181


155

Aureobasidium pullulans

alpha glucuronidase (aguA)
AY495374


156

Aspergillus tubingensis

aguA gene
Y15405


157

Thermotoga maritima

aguA gene
Y09510


158

Trichoderma reesei

alpha-glucuronidase
Z68706


159

Cellvibrio japonicus

alpha-glucuronidase (glcA67A)
AY065638




gene


160

Cellvibrio mixtus

alpha-glucuronidase (glcA67A)
AY065639




gene


161

Bacillus

alpha-glucuronidase (aguA)
AF441188




stearothermophilus strain

gene



T-1


162

Bacillus

alpha-glucuronidase (aguA)
AF221859




stearothermophilus

gene


163

Bacillus sp. TS-3

abn-ts gene for arabinase-TS
AB061269


164

Bacillus subtilis

endo-arabinase
D85132


165

Aspergillus niger

endo-1,5-alpha-L-arabinase
L23430




(abnA) gene


166

Piromyces communis

endo-1,3-1,4-beta-glucanase
EU314936




(licWF3)


167

Neocallimastix

endo-1,3-1,4-beta-glucanase
EU314934




patriciarum

(lic6)


168

Bacillus subtilis

lichenase(1,3;1,4-B-D-Glucan
E01881




4-glucanohydrolase)


169

Clostridium

lichenase licB gene for 1,3-
X58392




thermocellum

(1,3:1,4)-beta-D-glucan 3(4)-




glucanohydrolase


170

Streptococcus equinus

beta-(1,3-1,4)-glucanase
Z92911


171

Bacillus subtilis

bglS gene for beta-1,3-1,4-
Z46862




glucanase


172

Bacillus sp.

bgaA gene for lichenase
Z12151


173

Clostridium

licB gene for beta-1,3-1,4-
X63355




thermocellum

glucanase


174

Bacillus circulans

BGC gene for lichenase
X52880


175

Anaeromyces sp. W-98

lichenase (licB)
AF529296


176

Bacillus licheniformis

lichenase gene
AY383603



KCCM41412


177

Orpinomyces sp. PC-2

lichenase (licA)
U63813


178

Phanerochaete

endo-1,4-beta-D-mannanase
DQ779964




chrysosporium



179

Alicyclobacillus

endo-beta-1,4-mannanase gene
DQ680160




acidocaldarius



180

Clostridium

mannanase (man26A) and GH9
DQ778334




cellulolyticum H10

cellulase (cel9P) genes



GH26


181

Aspergillus sulphureus

beta-mannanase gene
DQ328335


182

Phanerochaete

Man5C
DQ779965




chrysosporium strain




RP78


183

Armillariella tabescens

mannanase
DQ286392


184

Agaricus bisporus

cel4 gene for CEL4a
AJ271862




mannanase


185

Bacillus sp. JAMB750

man26A gene for mannanase
AB128831


186

Clostridium

man26B gene for mannanase
AB044406




thermocellum

26B


187

Bacillus circulans isolate

mannanase gene
AY913796



Y203


188

Bacillus subtilis strain Z-2

mannose-6-phosphate
AY827489




isomerase and beta-1,4-




mannanase genes


189

Bacillus subtilis strain

beta-mannanase (man) gene
DQ269473



A33


190

Bacillus subtilis

mannanase gene
DQ351940


191

Bacillus circulans isolate

mannanase (man1) gene
AY907668



196


192

Bacillus sp. JAMB-602

amn5A gene for mannanase
AB119999


193

Agaricus bisporus

mannanase CEL4b (cel4 gene)
Z50095


194

Piromyces sp.

endo-b1,4-mannanase
X97520


195

Piromyces sp.

endo-1,4 beta-mannanase
X97408


196

Piromyces sp.

mannanase A
X91857


197

Clostridium

manA gene for mannanase A
AJ242666




thermocellum



198

Paecilomyces lilacinus

beta-1,3-mannanase
AB104400


199

Bacillus circulans

mannanase gene
AY623903


200

Bacillus circulans

mannanase gene
AY540747


201

Dictyoglomus

beta-mannanase (manA) gene
AF013989




thermophilum



202

Cellvibrio japonicus

manA gene
X82179


203

Bacillus circulans

aman6 gene for alpha-1,6-
AB024331




mannanase


204

Bacillus

beta-1,4-mannanase (manF),
AF038547




stearothermophilus

esterase (estA), and alpha-




galactosidase (galA) genes


205

Orpinomyces sp. PC-2

mannanase ManA (manA)
AF177206


206

Bacillus subtilis

mannose-6-phosphate
AF324506




isomerase and endo-1,4-beta-




mannosidase genes


207

Rhodothermus marinus

manA gene
X90947


208

Bacillus subtilis

gene for beta-mannanase
D37964


209

Thermotoga maritima

manA gene
Y17982


210

Cellulomonas fimi

Man26A (man26A) gene
AF126471


211

Streptomyces lividans

mannanase (manA) gene
M92297


212

Caldicellulosiruptor

beta-1,4-mannanase (manA)
U39812




saccharolyticus

gene


213

Bacillus sp.

beta-mannanase
AB016163


214

Bacillus circulans

mannanase
AB007123


215

Caldicellulosiruptor

beta-D-mannanase (manA)
M36063




saccharolyticus



216

Caldocellum

beta-mannanase/endoglucanase
L01257




saccharolyticum

(manA)


217

Aspergillus aculeatus

mannanase (man1)
L35487


218

Trichoderma reesei

beta-mannanase
L25310


219

Bacillus sp.

beta-mannanase gene
M31797


220

Aspergillus niger CBS

beta-mannosidase mndA
XM_001394595



513.88


221

Aspergillus niger CBS

beta-galactosidase lacA
XM_001389585



513.88


222

Aspergillus clavatus

beta-mannosidase
XM_001268087



NRRL 1


223

Neosartorya fischeri

beta-galactosidase
XM_001259270



NRRL 181


224

Neosartorya fischeri

beta-galactosidase
XM_001259223



NRRL 181


225

Aspergillus niger

mndA gene for beta-
AJ251874




mannosidase


226

Aspergillus niger

aglC gene for alpha-
AJ251873




galactosidase C


227

Aspergillus terreus

beta-glucuronidase
XM_001218602



NIH2624


228

Emericella nidulans

beta-mannosidase
DQ490488


229

Thermotoga neopolitana

manA gene
Y17983


230

Thermotoga neopolitana

manB gene
Y17981


231

Thermotoga maritima

manB gene
Y17980


232

Thermobifida fusca

manB gene for beta-D-
AJ489440




mannosidase


233

Thermotoga maritima

manA gene
Y17982


234

Pyrococcus furiosus

beta-mannosidase (bmnA) gene
U60214


235

Aspergillus aculeatus

beta-mannosidase
AB015509


236

Bacteroides fragilis

glaB gene for alpha-
AM109955




galactosidase


237

Bacteroides fragilis

glaA gene for alpha-
AM109954




galactosidase


238

Aspergillus fumigatus

alpha-galactosidase
XM_744777



Af293


239

Aspergillus fumigatus

alpha-galactosidase
XM_743036



Af293


240

Aspergillus niger CBS

extracellular alpha-glucosidase
XM_001402016



513.88
aglU


241

Aspergillus niger CBS

alpha-galactosidase aglA-
XM_001390808



513.88

Aspergillus niger (aglA)



242

Aspergillus niger CBS

alpha-galactosidase aglB
XM_001400207



513.88


243

Bacteroides

glaB gene for alpha-
AM109957




thetaiotaomicron

galactosidase


244

Bacteroides

glaA gene for alpha-
AM109956




thetaiotaomicron

galactosidase


245

Streptomyces avermitilis

gla gene for alpha-
AM109953



MA-4680
galactosidase


246

Aspergillus clavatus

alpha-galactosidase
XM_001276326



NRRL 1


247

Bifidobacterium longum

alpha-galactosidase (aglL)
AF160969


248

Neosartorya fischeri

alpha-galactosidase
XM_001266319



NRRL 181


249

Aspergillus niger

aglC gene for alpha-
AJ251873




galactosidase C


250

Aspergillus niger

aglB gene
Y18586


251

Lactobacillus fermentum

alpha-galactosidase (melA)
AY612895



strain CRL722
gene


252

Emericella nidulans

alpha-galactosidase (AN8138-
DQ490515




2)


253

Emericella nidulans

alpha-galactosidase
DQ490505


254

Pseudoalteromonas sp.

alpha-galactosidase gene
DQ530422



KMM 701


255

Lactobacillus plantarum

melA gene for alpha-
AJ888516




galactosidase


256

Bifidobacterium bifidum

alpha-galactosidase (melA)
DQ438978




gene


257

Lachancea

MELth2 gene for alpha-
AB257564




thermotolerans

galactosidase


258

Lachancea

MELth1 gene for alpha-
AB257563




thermotolerans

galactosidase


259

Clostridium stercorarium

Thermostable ALPHA-
BD359178




galactosidase gene


260

Bifidobacterium breve

alpha-galactosidase (aga2)
DQ267828



strain 203
gene


261

Clostridium josui

agaA gene for alpha-
AB025362




galactosidase


262

Trichoderma reesei

alpha-galactosidase
Z69253


263

Saccharomyces mikatae

alpha-galactosidase MEL gene
X95506


264

Saccharomyces

alpha-galactosidase MEL gene
X95505




paradoxus



265

Saccharomyces

alpha-galactosidase
Z37510




cerevisiae



266

Saccharomyces

alpha-galactosidase
Z37511




cerevisiae



267

Saccharomyces

alpha-galactosidase
Z37508




cerevisiae



268

Saccharomyces

MEL1 gene for alpha-
X03102




cerevisiae

galactosidase


269

Penicillium

alpha-galactosidase 1
AJ009956




simplicissimum



270

Clostridium stercorarium

aga36A gene for alpha-
AB089353




galactosidase


271

Bifidobacterium breve

alpha-galactosidase (aga) gene
AF406640


272

Lactobacillus plantarum

alpha-galactosidase (melA)
AF189765




gene


273

Mycocladus

Thermostable alpha-
BD082887 to




corymbiferus

galactosidase
BD082889


274

Bacillus

alpha-galactosidase AgaB
AY013287




stearothermophilus

(agaB)


275

Bacillus

alpha-galactosidase AgaA
AY013286




stearothermophilus

(agaA)


276

Bacillus

alpha-galactosidase AgaN
AF130985




stearothermophilus

(agaN)


277

Carnobacterium

AgaA (agaA)
AF376480




piscicola



278

Thermus sp. T2

alpha galactosidase
AB018548


279

Phanerochaete

alpha-galactosidase (agal) gene
AF246263




chrysosporium



280

Phanerochaete

alpha-galactosidase (agal) gene
AF246262




chrysosporium



281

Zygosaccharomyces

MELr gene for alpha-
AB030209




mrakii

galactosidase


282

Thermus thermophilus

beta-glycosidase (bglT) gene
AF135400



strain TH125


283

Torulaspora delbrueckii

MELt gene for alpha-
AB027130




galactosidase


284

Penicillium

alpha-galactosidase
AB008367




purporogenum



285

Mortierella vinacea

alpha-galactosidase
AB018691


286

Thermotoga neapolitana

alpha-1,6-galactosidase (aglA)
AF011400




gene


287

Trichoderma reesei

alpha-galactosidase
Z69254


288

Trichoderma reesei

alpha-galactosidase
Z69255




















TABLE III





No
Nucleotide ID
Gene name
Enzyme class
Species



















1
AM397952
lip3
EC 1.11.1.14

Phlebia tremellosa




cDNA


2
AM397951
lip2
EC 1.11.1.14

Phlebia tremellosa




cDNA


3
AJ745879
mnp
EC 1.11.1.13

Trametes versicolor




cDNA


4
AJ745080
mrp
EC 1.11.1.13

Trametes versicolor




cDNA


5
AY836676
mnp5
EC 1.11.1.13

Pleurotus pulmonarius




cDNA


6
AJ315701
mnp2
EC 1.11.1.13

Phlebia radiate




cDNA


7
AJ310930
mnp3
EC 1.11.1.13

Phlebia radiate




cDNA


8
AB191466
tclip
EC 1.11.1.14

Trametes cervina




cDNA


9
M24082
Lig1
EC 1.11.1.14

Phanerochaete chrysosporium



10
J04980
mp-1
EC 1.11.1.13

Phanerochaete chrysosporium




cDNA


11
M80213 M36814
lip
EC 1.11.1.14

Phanerochaete chrysosporium




cDNA


12
AF074951
cdh
EC 1.1.99.18

Corynascus heterothallicus




cDNA


13
X97832
cdh
EC 1.1.99.18

Phanerochaete chrysosporium



14
X88897
cdh
EC 1.1.99.18

Phanerochaete chrysosporium




cDNA


15
U50409
Cdh-1
EC 1.1.99.18

Phanerochaete chrysosporium



16
U65888
Cdh-2
EC 1.1.99.18

Phanerochaete chrysosporium



17
U46081
cdh
EC 1.1.99.18

Phanerochaete chrysosporium




D90341
celCCD
3.2.1.4

Clostridium cellulolyticum




AY339624
EglA


Bacillus pumilus




D83704
celJ, celK


Clostridium thermocellum




EF371844
egl


Ralstonia solanacearum




EF371842
egl


Ralstonia solanacearum




L02544
cenD
EC.3.2.1.4

Cellulomonas fimi




EU055604
cel9B


Fibrobacter succinogenes




EF093188
ega


Bacillus sp. AC-1




EF620915
endoglucanase


Bacillus pumilus




AB167732
egl


Paenibacillus sp. KSM-N659




AB167731
egl


Paenibacillus sp. KSM-N440




AB167730
egl


Paenibacillus sp.




AB167729
egl


Paenibacillus sp.




EF205153
endoglucanase


Thermomonospora sp. MTCC 5117




DQ657652
egl


Ralstonia solanacearum strain UW486




AJ616005
celA


Bacillus licheniformis




DQ294349
eglA


Azoarcus sp. BH72




DQ923327
eg1


uncultured Butyrivibrio sp




Z12157
cela1


Streptomyces halstedii




AJ275974
celI


Clostridium thermocellum




AB179780
cel5A


Eubacterium cellulosolvens




DQ176867
celK


Pectobacterium carotovorum (Erwinia








carotovora)




X57858
cenC


Cellulomonas fimi




AY298814
cel5B


Thermobifida fusca




X79241
celV1


Pectobacterium carotovorum




AY646113
engO


Clostridium cellulovorans




AB028320
egV


Ruminococcus albus




AB016777
egIV


Ruminococcus albus




X76640
celA


Myxococcus xanthus




Y12512
celA


Bacillus sp. BP-23




Z83304
endA


Ruminococcus flavefaciens




Z86104
celB, celC


Anaerocellum thermophilum




Z77855
celD


Anaerocellum thermophilum




X76000
celV


Pectobacterium carotovorum (Erwinia








carotovora)




X73953
eglS


Streptomyces rochei




X54932
celB


Ruminococcus albus




X54931
celA


Ruminococcus albus




X52615
endoglucanase


Cellvibrio japonicus




X69390
celG


Clostridium thermocellum




X03592
celB


Clostridium thermocellum




X17538
end1


Butyrivibrio fibrisolvens




AJ308623
celA


Alicyclobacillus acidocaldarius




AJ304415
engXCA


Xanthomonas campestris pv. campestris




AJ133614
celB


Bacillus sp. BP-23




AY445620
cel9A


Bacillus licheniformis




AF025769
celB


Erwinia carotovora subsp. carotovora




L20093
E4


Thermomonospora fusca




AJ551527
celB


Alicyclobacillus acidocaldarius




M64363
celF


Clostridium thermocellum




AB044407
celT


Clostridium thermocellum




AB059267
egl257


Bacillus circulans




AF363635
engA


Bacillus amyloliquefaciens




M31311
eglA


Clostridium saccharobutylicum




AF109242
celZ


Erwinia chrysanthemi




M31311
eglA


Clostridium saccharobutylicum




AF033262
celA


Pseudomonas sp. YD-15




M84963
endoglucanase


Bacillus subtilis




AB047845
celQ


Clostridium thermocellum




AY007311
celA


Clavibacter michiganensis subsp.








sepedonicus




AF132735
engK


Clostridium cellulovorans




U34793
engH


Clostridium cellulovorans




AF206716
endoglucanase


Bacillus pumilus




AF113404
cel6A


Cellulomonas pachnodae




X04584
celD


Clostridium thermocellum




U51222
celA2


Streptomyces halstedii




U27084
cel


Bacillus sp




L02868
celA


Clostridium longisporum




AF067428
Cel5A


Bacillus agaradhaerens




L01577
E3, E4, E5


Thermobifida fusca




M73321
E2


Thermobifida fusca




L20094
E1


Thermobifida fusca




U94825
Endoglucanase


Actinomyces sp. 40




U37056
engF


Clostridium cellulovoran




U33887
celG


Fibrobacter succinogenes




U08621
celB


Ruminococcus flavefaciens FD-1




Y00540
celZ


Erwinia chrysanthemi




U16308
celC


Caldocellum saccharolyticum




K03088
celA


Clostridium thermocellum




M93096
celCCA


Clostridium cellulolyticum




L03800
celE


Ruminococcus flavefaciens




L13461
celM


Clostridium thermocellum




M74044
celY


Erwina chrysanthemi




X13602
celB


Caldocellum saccharolyticum




AB078006
CBH II


Streptomyces sp. M23




X80993
cbhA


Clostridium thermocellum




AJ005783
cbhA, celK


Clostridium thermocellum




AY494547
cbhA


Clostridium thermocellum




AF039030
celK


Clostridium thermocellum




L38827
cbhB
EC 3.2.1.91

Cellulomonas fimi




L25809
cbhA


Cellulomonas fimi




EU314939
cbhYW23-4


Piromyces rhizinflatus




EU314933
cbh6


Neocallimastix patriciarum




EF397602
cbh1


Penicillium decumbens




AB298323
cel1, cel2


Polyporus arcularius




AB298322
cbh I


Polyporus arcularius




EU038070
cbh I


Fusicoccum sp. BCC4124




EF624464
cbh


Thermomyces lanuginosus




AM262873
cbhI-2


Pleurotus ostreatus




AM262872
cbhI-4


Pleurotus ostreatus




AM262871
cbhI-3


Pleurotus ostreatus




AM262993
cbhI-1


Pleurotus ostreatus




XM_745507
Cbh-celD


Aspergillus fumigatus




AY973993
exo-(cbhI)


Penicillium chrysogenum




AF421954
cbh


Thermoascus aurantiacus




XM_001389539
cbhB


Aspergillus niger




XM_001391971
cbhA


Aspergillus niger




DQ864992
CBHII


Trichoderma viride




EF222284
cbh3


Chaetomium thermophilum




X69976
cbh1


Hypocrea koningii/Trichoderma koningii




Z29653
exo-cbhI.2


Phanerochaete chrysosporium




Z22527
exo-cbhI


Phanerochaete chrysosporium




X53931
cbh


Trichoderma viride




X54411
Pccbh1-1


Phanerochaete chrysosporium




DQ085790
cbh3


Chaetomium thermophilum




AY559104
cbhII-I


Volvariella volvacea




AY559102
cbhI-I


Volvariella volvacea




D86235
cbh1


Trichoderma reesei




DQ504304
cbhII


Hypocrea koningii strain 3.2774




E00389
cbh


Hypocrea jecorina/Trichoderma reesei




AY706933
cbh-C


Gibberella zeae




AY706932
cbh-C


Fusarium venenatum




AY706931
cbh-C


Gibberella zeae




DQ020255
cbh-6


Chaetomium thermophilum




AY954039
cbh


Schizophyllum commune




D63515
cbh-1


Humicola grisea var. thermoidea




Z50094
cel2-cbh


Agaricus bisporus




Z50094
Exocellobiohydrolase


Agaricus bisporus




Z22528
exo-cbh I


Phanerochaete chrysosporium




AY840982
cbh


Thermoascus aurantiacus var. levisporus




AY761091
cbhII


Trichoderma parceramosum




AY651786
cbhII


Trichoderma parceramosum




AY690482
cbhI


Penicillium occitanis




CQ838174
cbh


Malbranchea cinnamomea




CQ838172
cbh


Stilbella annulata




CQ838150
cbh


Chaetomium thermophilum




AY328465
celB-cbh


Neocallimastix frontalis




AB177377
cexI, cbh


Irpex lacteus




AY531611
cbh-I


Trichoderma asperellum




AY116307
cbh-7


Cochliobolus heterostrophus




AB002821
cbh-I


Aspergillus aculeatus




AF478686
cbh-1


Thermoascus aurantiacus




AY368688
cbh-II


Trichoderma viride strain CICC 13038




AY368686
cbhI


Trichoderma viride




AY091597
Cel6E


Piromyces sp. E2




AX657625
cbh


Phanerochaete chrysosporium




AX657629
cbh


Aspergillus sp.




AX657633
cbh


Pseudoplectania nigrella




U97154
celF


Orpinomyces sp. PC-2




U97152
celD-cbh


Orpinomyces sp. PC-2




AF439935
cbh1A


Talaromyces emersonii




AB021656
cbhI


Trichoderma viride




AB089343
cbh


Geotrichum sp. M128




AB089436
celC-cbh


Aspergillus oryzae




A35269
cbh


Fusarium oxysporum




AF439936
cbhII


Talaromyces emersonii




AY075018
cbhII


Talaromyces emersonii




AY081766
cbh1


Talaromyces emersonii




AF378175
cbh1


Trichoderma koningii




AF378173
cbh2


Trichoderma koningii




AF378174
cbh2


Trichoderma koningii




AF244369
cbhII-1


Lentinula edodes




L22656
cbh1-4


Phanerochaete chrysosporium




L24520
cel3AC-cbh


Agaricus bisporus




M22220
cbhI


Phanerochaete chrysosporium




AY050518
cbh-II


Pleurotus sajor-caju




AF223252
cbh-1


Trichoderma harzianum




AF177205
celI-cbh


Orpinomyces sp. PC-2




AF177204
celH-cbh


Orpinomyces sp. PC-2




AF302657
cbh-II


Hypocrea jecorina




AF156269
cbh-2


Aspergillus niger




AF156268
cbh-2


Aspergillus niger




AF123441
cbh1.2


Humicola grisea var. thermoidea




U50594
cbh1.2


Humicola grisea




M55080
cbh-II


Trichoderma reesei




M16190
cbh-II


Trichoderma reesei



1
NC_000961
endoglucanase
EC 3.1.2.4

Pyrococcus horikoshii



2
NC_000961/U33212/
cel5A
EC 3.1.2.4

Acidothermus cellulolyticus 11B; ATCC




AX467594


43068


3
M32362
cel5A
EC 3.1.2.4

Clostridium cellulolyticum



4
M22759
celE, cel5C
EC 3.2.1.4

Clostridium thermocellum



5
AJ307315
celC
EC 3.2.1.4

Clostridium thermocellum



6
AJ275975
celO
EC 3.2.1.91

Clostridium thermocellum



7
X03592
celB/cel5A
EC 3.2.1.4

Clostridium thermocellum



8
Z29076
eglS
EC 3.2.1.4

Bacillus subtilis



9
M33762
celB
EC 3.2.1.4

Bacillus lautus (strain PL236)



10
L25809
cbhA
EC 3.2.1.91

Cellulomonas fimi



11
M15823
cenA/cel6A
EC 3.2.1.4

Cellulomonas fimi



12
M73321
cel6A
EC 3.2.1.4

Thermobifida fusca



13
U18978
cel6B
EC 3.2.1.91

Thermomonospora fusca



14
X65527
cellodextrinase D
EC 3.2.1.74

Cellvibrio japonicus



15
L06134
ggh-A
EC 3.2.1.74

Thermobispora bispora



16
U35425
cdxA
EC 3.2.1.74

Prevotella bryantii



17
EU352748
cel9D
EC 3.2.1.74

Fibrobacter succinogenes



18
AAL80566
bglB
EC 3.2.1.21

Pyrococcus furiosus



19
Z70242
bglT
EC 3.2.1.21

Thermococcus sp



20
M96979
bglA
EC 3.2.1.21

Bacillus circulans



21
AB009410
beta-glucosidase
EC 3.2.1.21

Bacillus sp. GL1



22
D88311 D84489
beta-D-glucosidase
EC 3.2.1.21

Bifidobacterium breve



23
AAQ00997
BglA
EC 3.2.1.21

Clostridium cellulovorans



24
X15644
bglA
EC 3.2.1.21

Clostridium thermocellum



25
AAA25311
BglB
EC 3.2.1.21

Thermobispora bispora



26
AB198338
bglA
EC 3.2.1.21

Paenibacillus sp. HC1



27
AF305688
Bgl1
EC 3.2.1.21

Sphingomonas paucimobilis



28
CAA91220
beta-glucosidase
EC 3.2.1.21

Thermoanaerobacter brockii



29
CAA52276
beta-glucosidase
EC 3.2.1.21

Thermotoga maritime



30
AAO15361
beta-glucosidase
EC 3.2.1.21

Thermus caldophilus



31
Z97212
beta-glucosidase
EC 3.2.1.21

Thermotoga neapolitana



32
AB034947
beta-glucosidase
EC 3.2.1.21

Thermus sp. Z-1



33
M31120
beta-glucosidase
EC 3.2.1.21

Butyrivibrio fibrisolvens



34
D14068
beta-glucosidase
EC 3.2.1.21

Cellvibrio gilvus



35
AF015915
Bg1
EC 3.2.1.21

Flavobacterium meningosepticum



36
U08606
bgxA
EC 3.2.1.21

Erwinia chrysanthemi



37
Z94045
bglZ
EC 3.2.1.21

Clostridium stercorarium



38
AY923831
bglY
EC 3.2.1.21

Paenibacillus sp.



38
Z56279
xglS
EC 3.2.1.21

Thermoanaerobacter brockii



39
CQ893499
bglB
EC 3.2.1.21

Thermotoga maritime




DQ916114
beta-glucosidase
EC 3.2.1.21
uncultured bacterium




(RG11)



DQ182493
beta-glucosidase
EC 3.2.1.21
uncultured bacterium



DQ916117
beta-glucosidase
EC 3.2.1.21
uncultured bacterium



DQ022614
umbgl3A
EC 3.2.1.21
uncultured bacterium



DQ916115
RG12 beta-
EC 3.2.1.21
uncultured bacterium




glucosidase gene



DQ182494
beta-glucosidase
EC 3.2.1.21
uncultured bacterium




(umbgl3C)



DQ916118
Uncultured bacterium
EC 3.2.1.21
uncultured bacterium




clone RG25



DQ916116
RG14 beta-
EC 3.2.1.21
uncultured bacterium




glucosidase gene



U12011
Bg1
EC 3.2.1.21
unidentified bacterium



AB253327
bgl1B
EC 3.2.1.21

Phanerochaete chrysosporium




AB253326
beta-
EC 3.2.1.21

Phanerochaete chrysosporium





glucosidase



AJ276438
beta-
EC 3.2.1.21

Piromyces sp. E2





glucosidase



AF439322
bg1
EC 3.2.1.21

Talaromyces emersonii




D64088 cDNA
beta-glucosidase 1
EC 3.2.1.21

Aspergillus aculeatus




DQ490467
beta-glucosidase 1
EC 3.2.1.21

Emericella nidulans




cDNA



AAB27405
beta-glucosidase
EC 3.2.1.21

Aspergillus niger




AX616738
beta-glucosidase
EC 3.2.1.21

Aspergillus oryzae




AJ130890

EC 3.2.1.21

Botryotinia fuckeliana




L21014
beta-glucosidase
EC 3.2.1.21

Dictyostelium discoideum




U87805
bgl1
EC 3.2.1.21

Coccidioides posadasii




AM922334
beta-glucosidase
EC 3.2.1.21

Rhizomucor miehei




DQ114396
bgl1
EC 3.2.1.21

Thermoascus aurantiacus




CS497644
beta-glucosidase
EC 3.2.1.21

Penicillium brasilianum




DD463307
beta-glucosidase
EC 3.2.1.21

Aspergillus oryzae




DD463306
beta-glucosidase
EC 3.2.1.21

Aspergillus oryzae




DD463305
beta-glucosidase
EC 3.2.1.21

Aspergillus oryzae




DQ926702
beta-glucosidase
EC 3.2.1.21

Rhizoctonia solani




AY281378
beta-glucosidase
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei





cel3D



EU029950
beta-glucosidase
EC 3.2.1.21

Penicillium occitanis




EF648280
beta-glucosidase
EC 3.2.1.21

Chaetomium thermophilum




EF527403
bgl1
EC 3.2.1.21

Penicillium brasilianum




DQ114397
bgl1
EC 3.2.1.21

Thermoascus aurantiacus




XM_001398779
bgl1
EC 3.2.1.21

Aspergillus niger




XM_001274044
beta-glucosidase
EC 3.2.1.21

Aspergillus clavatus




AF121777
beta-glucosidase
EC 3.2.1.21

Aspergillus niger




AJ566365
beta-glucosidase
EC 3.2.1.21

Aspergillus oryzae




AJ132386
bgl1
EC 3.2.1.21

Aspergillus niger




AF016864
beta-glucosidase
EC 3.2.1.21

Orpinomyces sp. PC-2




CS435985
beta-glucosidase
EC 3.2.1.21

Hypocrea jecorina




DQ011524
bgl2
EC 3.2.1.21

Thermoascus aurantiacus




DQ011523
bgl2
EC 3.2.1.21

Thermoascus aurantiacus




DD329362
Bgl6
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




DD329363
Bgl6
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




DQ888228
bgl
EC 3.2.1.21

Chaetomium thermophilum




DQ655704
bgl
EC 3.2.1.21

Aspergillus niger




DQ010948
bgl
EC 3.2.1.21

Pichia anomala/Candida beverwijkiae




DQ010947
beta-glucosidase
EC 3.2.1.21

Hanseniaspora uvarum




DD146974
Bgl
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




DD146973
Bgl
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




DD182179
Bgl
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




DD182178
Bgl
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




DD181296
Bgl
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




DD181295
Bgl
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




CS103208
beta-glucosidase
EC 3.2.1.21

Aspergillus fumigatus




AJ276438
bgl1A
EC 3.2.1.21

Piromyces sp. E2




AY943971
bgl1
EC 3.2.1.21

Aspergillus avenaceus




AY688371
bgl1
EC 3.2.1.21

Phaeosphaeria avenaria




AY683619
bgl1
EC 3.2.1.21

Phaeosphaeria nodorum




AB081121
beta-glucosidase
EC 3.2.1.21

Phanerochaete chrysosporium




AY445049
bgla
EC 3.2.1.21

Candida albicans




AF500792
beta-glucosidase
EC 3.2.1.21

Piromyces sp. E2




AY343988
beta-glucosidase
EC 3.2.1.21

Trichoderma viride




AY072918
beta-glucosidase
EC 3.2.1.21

Talaromyces emersonii




BD185278
beta-glucosidase
EC 3.2.1.21

Debaryomyces hansenii/Candida famata




BD178410
beta-glucosidase
EC 3.2.1.21

Debaryomyces hansenii/Candida famata




BD168028
beta-glucosidase
EC 3.2.1.21

Acremonium cellulolyticus




AB003110
bgl
EC 3.2.1.21

Hypocrea jecorina/Trichoderma reesei




AB003109
bgl4
EC 3.2.1.21

Humicola grisea var. thermoidea




AY049946
BGL5
EC 3.2.1.21

Coccidioides posadasii




AF338243
BGL3
EC 3.2.1.21

Coccidioides posadasii




AY081764
beta-glucosidase
EC 3.2.1.21

Talaromyces emersonii




AY049947
BGL6
EC 3.2.1.21

Coccidioides posadasii




AY049945
BGL4
EC 3.2.1.21

Coccidioides posadasii




AY049944
BGL3
EC 3.2.1.21

Coccidioides posadasii




AF022893
BGL2
EC 3.2.1.21

Coccidioides posadasii




AX011537
beta-glucosidase
EC 3.2.1.21

Aspergillus oryzae




AF268911
beta-glucosidase
EC 3.2.1.21

Aspergillus niger




U31091
beta-glucosidase
EC 3.2.1.21

Candida wickerhamii




U13672
beta-glucosidase
EC 3.2.1.21

Candida wickerhamii




AF036873
beta-glucosidase
EC 3.2.1.21

Phanerochaete chrysosporium




AF036872
beta-glucosidase
EC 3.2.1.21

Phanerochaete chrysosporium




X05918
beta-glucosidase
EC 3.2.1.21

Kluyveromyces marxianus




U16259
beta-glucosidase
EC 3.2.1.21

Pichia capsulata





(bgln)



M22476
BGL2


Saccharomycopsis fibuligera




M22475
BGL1


Saccharomycopsis fibuligera




M27313
beta-glucosidase


Schizophyllum commune




















TABLE IV





No.
Source
Gene
Accession No.


















1

Gibberella zeae

triacylglycerol lipase FGL5
EU402385


2

Schizosaccharomyces pombe

972h-triacylglycerol lipase
NM_001023305




(SPCC1450.16c)


3

Schizosaccharomyces pombe

972h-esterase/lipase (SPAC8F11.08c)
NM_001019384


4

Schizosaccharomyces pombe

972h-triacylglycerol lipase
NM_001018593




(SPAC1A6.05c)


5

Hypocrea lixii

lip1 gene for lipase 1
AM180877


6

Thermomyces lanuginosus

lipase (LGY) gene
EU022703


7

Aspergillus niger strain F044

triacylglycerol lipase precursor
DQ647700


8

Antrodia cinnamomea

lipase
EF088667


9

Aspergillus oryzae

tglA gene for triacylglycerol lipase
AB039325


10

Gibberella zeae

triacylglycerol lipase FGL4
EU191903


11

Gibberella zeae

triacylglycerol lipase FGL2
EU191902


12

Gibberella zeae

triacylglycerol lipase (fgl3)
EU139432


13

Aspergillus tamarii isolate FS132

lipase
EU131679


14

Aureobasidium pullulans strain

extracellular lipase gene
EU117184



HN2.3


15

Rhizopus microsporus var.

lipase
EF405962



chinensis


16

Fusarium oxysporum

lipase (lip1)
EF613329


17

Aspergillus tamarii isolate FS132

lipase
EF198417


18

Neosartorya fischeri NRRL 181

Secretory lipase (NFIA_072820)
XM_001259263


19

Neosartorya fischeri NRRL 181

Secretory lipase (NFIA_047420)
XM_001257303


20

Nectria haematococca

NhL1 gene for extracellular lipase
AJ271094


21

Aspergillus terreus NIH2624

lipase precursor (ATEG_09822)
XM_001218443


22

Galactomyces geotrichum

lipase
DQ841229


23

Yarrowia lipolytica

lipase 2
DQ831123


24

Magnaporthe grisea

vacuolar triacylglycerol lipase (VTL1)
DQ787100


25

Magnaporthe grisea

triacylglycerol lipase (TGL3-2)
DQ787099


26

Magnaporthe grisea

triacylglycerol lipase (TGL3-1)
DQ787098


27

Magnaporthe grisea

triacylglycerol lipase (TGL2)
DQ787097


28

Magnaporthe grisea

triacylglycerol lipase (TGL1-2)
DQ787096


29

Magnaporthe grisea

triacylglycerol lipase (TGL1-1)
DQ787095


30

Magnaporthe grisea

hormone-sensitive lipase (HDL2)
DQ787092


31

Magnaporthe grisea

hormone-sensitive lipase (HDL1)
DQ787091


32

Penicillium expansum

triacylglycerol lipase
DQ677520


33

Aspergillus niger

triacylglycerol lipase B (lipB)
DQ680031


34

Aspergillus niger

triacylglycerol lipase A (lipA)
DQ680030


35

Yarrowia lipolytica

lip2
AJ012632


36

Galactomyces geotrichum

triacylglycerol lipase
X81656


37

Candida antarctica

lipase B
Z30645


38

Candida cylindracea

LIP1 to LIP5 gene for lipase
X64703 to





X64708


39

Yarrowia lipolytica

LIPY8p (LIPYS) gene
DQ200800


40

Yarrowia lipolytica

LIPY7p (LIPY7) gene
DQ200799


41

Rhizopus niveus

prepro thermostable lipase
E12853


42

Galactomyces geotrichum

lipase
E02497


43

Rhizomucor miehei

lipase
A02536


44


45

Fusarium heterosporum

lipase
S77816


46

Candida albicans SC5314

triglyceride lipase (CaO19_10561)
XM_716177


47

Candida albicans SC5314

triglyceride lipase (CaO19_3043)
XM_716448


48

Rhizopus stolonifer

lipase lipRs
DQ139862


49

Candida albicans

secretory lipase 3 (LIP3) gene to lipase
AF191316 to




10 (LIP10)
AF191323


50

Candida albicans

secretory lipase 1 (LIP1)
AF188894


51

Candida albicans

secretory lipase (LIP2) gene
AF189152


52

Emericella nidulans

triacylglycerol lipase (lipA) gene
AF424740


53

Gibberella zeae

extracellular lipase (FGL1)
AY292529


54

Kurtzmanomyces sp. I-11

lipase
AB073866


55

Candida deformans

lip1 gene to lip 3 for triacylglycerol
AJ428393 to




lipase
AJ428395


56

Botryotinia fuckeliana

lipase (lip1)
AY738714


57

Penicillium allii

lipase (lipPA)
AY303124


58

Aspergillus flavus

lipase
AF404489


59

Aspergillus parasiticus

lipase
AF404488


60

Yarrowia lipolytica

LIP4 gene for lipase
AJ549517


61

Candida parapsilosis

lip1 gene for lipase 1 and lip2 gene for
AJ320260




lipase 2


62

Penicillium expansum

triacylglycerol lipase precursor
AF288685


63

Penicillium cyclopium

alkaline lipase
AF274320


64

Pseudomonas sp.

lip35 lipase gene
EU414288


65

Bacillus sp. NK13

lipase gene
EU381317


66

Uncultured bacterium

lipase/esterase gene
EF213583 to





EF213587


67

Shewanella piezotolerans

WP3 lipase gene
EU352804


68

Bacillus sp. Tosh

lipase (lipA) gene
AY095262


69

Bacillus subtilis strain FS321

lipase
EF567418


70

Pseudomonas fluorescens strain

lipase
EU310372



JCM5963


71

Pseudomonas fluorescens

triacylglycerol lipase
D11455


72

Burkholderia cepacia

alkaline lipase
EU280313


73

Burkholderia sp. HY-10

lipase (lipA) and lipase foldase (lifA)
EF562602




genes


74

Pseudomonas aeruginosa

lip9, lif9 genes for LST-03 lipase, lipase-
AB290342




specific foldase


75

Pseudomonas fluorescens

gene for lipase
AB009011


76

Streptomyces fradiae

clone k11 lipase gene
EF429087


77

Geobacillus zalihae strain T1

thermostable lipase gene
AY260764


78

Pseudomonas sp. MIS38

gene for lipase
AB025596


79

Uncultured bacterium

cold-active lipase (lipCE) gene
DQ925372


80

Burkholderia cepacia

lipase (lipA) and lipase chaperone (lipB)
DQ078752




genes


81

Psychrobacter sp.

2-17 lipase gene
EF599123


82

Bacillus subtilis strain Fs32b

lipase gene
EF541144


83

Bacillus subtilis strain FS14-3a

lipase gene
EF538417


84

Aeromonas hydrophila strain J-1

extracellular lipase gene
EF522105


85

Acinetobacter sp. MBDD-4

lipase gene
DQ906143


86

Photorhabdus luminescens subsp.

lipase 1 (lip1) gene
EF213027




akhurstii strain 1007-2



87

Uncultured bacterium clone

lipase gene
DQ118648



h1Lip1


88

Geobacillus thermoleovorans

lipase precursor (lipA) gene
EF123044


89

Bacillus pumilus strain F3

lipase precursor
EF093106


90

Pseudomonas fluorescens lipase

lipase (lipB68) gene
AY694785



(lipB68) gene


91

Geobacillus stearothermophilus

thermostable lipase precursor gene
EF042975



strain ARM1


92

Serratia marcescens strain

extracellular lipase (lipA) gene
DQ884880



ECU1010


93

Bacillus subtilis

lipase gene
DQ250714


94

Pseudomonas aeruginosa

gene for lipase modulator protein
D50588


95

Pseudomonas aeruginosa

gene for lipase
D50587


96

Uncultured bacterium clone pUE5

esterase/lipase (estE5) gene
DQ842023


97

Serratia marcescens strain ES-2

lipase (esf) gene
DQ841349


98

Pseudomonas fluorescens strain

lipase class 3 gene
DQ789596



26-2


99

Stenotrophomonas maltophilia

lipase gene
DQ647508



strain 0450


100

Listonella anguillarum

Plp (plp), Vah1 (vah1), LlpA (llpA), and
DQ008059




LlpB (llpB) genes


101

Geobacillus sp.

SF1 lipase gene
DQ009618


102

Uncultured Pseudomonas sp.

lipase (lipJ03) gene
AY700013


103

Photobacterium sp. M37

lipase gene
AY527197


104

Pseudomonas fluorescens

lipase (lip) gene
DQ305493


105

Pseudomonas aeruginosa

lipase (lipB) gene
DQ348076


106

Bacillus pumilus mutant

lipase precursor
DQ345448


107

Bacillus pumilus strain YZ02

lipase gene
DQ339137


108

Geobacillus thermoleovorans YN

thermostable lipase (lipA) gene
DQ298518


109

Pseudomonas sp. CL-61

lipase (lipP) gene
DQ309423


110

Burkholderia sp. 99-2-1

lipase (lipA) gene
AY772174


111

Burkholderia sp. MC16-3

lipase (lipA) gene
AY772173


112

Pseudomonas fluorescens

lipase (lipB52) gene
AY623009


113

Geobacillus stearothermophilus

lipase gene
AY786185


114

Burkholderia multivorans strain

LifB (lifB) gene
DQ103702



Uwc 10


115

Burkholderia multivorans strain

LipA (lipA) gene
DQ103701



Uwc 10


116

Bacillus pumilus

lipase precursor gene
AY494714


117

Bacillus sp. TP10A.1

triacylglycerol lipase (lip1) gene
AF141874


118

Staphylococcus warneri

gehWC gene for lipase
AB189474


119

Staphylococcus warneri

lipWY gene for lipase
AB189473


120

Bacillus sp. L2

thermostable lipase gene
AY855077


121

Bacillus megaterium

lipase/esterase gene
AF514856


122

Pseudomonas aeruginosa

lip8 gene for lipase
AB126049


123

Bacillus sp. 42

thermostable organic solvent tolerant
AY787835




lipase gene


124

Pseudomonas fluorescens

lipase (lipB41) gene
AY721617


125

Burkholderia cepacia

triacylglycerol lipase (LipA) and lipase
AY682925




chaperone (LipB) genes


126

Pseudomonas aeruginosa

triacylglycerol lipase (LipA) and lipase
AY682924




chaperone (LipB) genes


127

Vibrio vulnificus

lipase and lipase activator protein genes
AF436892


128

Uncultured bacterium plasmid

lipase (lipA) gene
AF223645



pAH114


129

Thermoanaerobacter

lipase (lip1) gene
AY268957




tengcongensis



130

Pseudomonas aeruginosa

lip3 gene for lipase
AB125368


131

Staphylococcus epidermidis

lipase precursor (gehD) gene
AF090142


132

Pseudomonas fluorescens

lipA gene for lipase
AB109036



clone: pLP101-2741


133

Pseudomonas fluorescens

lipA gene for lipase
AB109035



clone: pLPM101


134

Pseudomonas fluorescens

lipA gene for lipase
AB109034



clone: pLPD101


135

Pseudomonas fluorescens

lipA gene for lipase
AB109033



clone: pLP101


136

Micrococcus sp. HL-2003

lipase gene
AY268069


137

Pseudomonas sp. JZ-2003

lipase gene
AY342316


138

Vibrio harveyi

vest gene
AF521299


139

Pseudomonas fluorescens

lipase gene
AY304500


140

Bacillus sphaericus strain 205y

lipase gene
AF453713


141

Bacillus subtilis

lipase (lipE) gene
AY261530


142

Synthetic construct

triacylglycerol lipase gene
AY238516


143

Serratia marcescens

lipA gene for lipase
D13253


144

Geobacillus thermoleovorans IHI-

thermophilic lipase gene
AY149997



91


145

Streptomyces rimosus

GDSL-lipase gene
AF394224


146

Pseudomonas luteola

triacylglycerol lipase precursor gene
AF050153


147

Geobacillus thermoleovorans

thermostable lipase (lipA)
AY095260


148

Mycoplasma hyopneumoniae

triacylglycerol lipase (lip) gene
AY090779


149

Staphylococcus aureus

lipase gene
AY028918


150

Bacillus sp. B26

lipase gene
AF232707


151

Pseudomonas aeruginosa

triacylglycerol acylhydrolase (lipA)
AF237723


156

Bacillus stearothermophilus

lipase gene
AF429311


157

Bacillus stearothermophilus

lipase gene
AF237623


158

Bacillus thermoleovorans

lipase (ARA) gene
AF134840


159

Bacillus stearothermophilus

lipase gene
U78785


160

Pseudomonas fluorescens

lipase (lipB) gene
AF307943


161

Pseudomonas sp. KB700A

KB-lip gene for lipase
AB063391


162

Moritella marina

super-integron triacylglycerol acyl
AF324946




hydrolase (lip) gene


163

Staphylococcus xylosus

lipase precursor GehM (gehM) gene
AF208229


164

Staphylococcus warneri

lipase precursor (gehA) gene
AF208033


165

Pseudomonas sp. UB48

lipase (lipUB48) gene
AF202538


166

Psychrobacter sp. St1

lipase (lip) gene
AF260707


167

Pseudomonas fluorescens

polyurethanase lipase A (pulA) gene
AF144089


168

Staphylococcus haemolyticus

lipase gene
AF096928


169

Pseudomonas fluorescens

genes for ABC exporter operon
AB015053


170

Pseudomonas aeruginosa

lipase (lipC) gene
U75975


171

Streptomyces coelicolor

lipase (lipA), and LipR activator (lipR)
AF009336




genes


172
Petroleum-degrading bacterium
gene for esterase HDE
AB029896



HD-1


173

Pseudomonas fragi

lipase precursor gene
M14604


174

Acinetobacter calcoaceticus

lipase (lipA) and lipase chaperone (lipB)
AF047691




genes


175

Streptomyces albus

lipase precursor (lip) and LipR genes
U03114


176

Staphylococcus epidermidis

lipase precursor (geh1) gene
AF053006


177

Pseudomonas sp. B11-1

lipase (lipP) gene
AF034088


178

Pseudomonas fluorescens

lipase (lipA) gene
AF031226


179

Pseudomonas aeruginosa

gene for lipase
AB008452


180

Pseudomonas wisconsinensis

extracellular lipase (lpwA) and lipase
U88907




helper protein (lpwB) genes


181

Aeromonas hydrophila

extracellular lipase (lip) gene
U63543


182

Streptomyces sp.

triacylglycerol acylhydrolase (lipA) and
M86351




lipA transcriptional activator (lipR) genes


183

Proteus vulgaris

alkaline lipase gene
U33845


184

Moraxella sp.

lip3 gene for lipase 3
X53869


185

Serratia marcescens SM6

extracellular lipase (lipA)
U11258


186

Staphylococcus aureus

geh gene encoding lipase (glycerol ester
M12715




hydrolase)


187

Pseudomonas fluorescens

lipase gene
M86350


188

Bacillus subtilis

lipase (lipA)
M74010


189

Staphylococcus epidermidis

lipase (gehC) gene
M95577



















TABLE V





Nucleotide Id
Gene
Enzyme class
Species







EU367969
alpha-amylase
3.2.1.1

Bacillus amyloliquefaciens



BD249244
Alpha-amylase


Bacillus amyloliquefaciens



DD238310
Alpha-amylase


Bacillus amyloliquefaciens



BD460864
Alpha-amylase


Bacillus amyloliquefaciens



V00092
Alpha-amylase


Bacillus amyloliquefaciens



M18424
Alpha-amylase


Bacillus amyloliquefaciens



J01542
Alpha-amylase


Bacillus amyloliquefaciens



EU184860
amyE


Bacillus subtilis



AM409180
amy


Bacillus subtilis



E01643
alpha-amylase


Bacillus subtilis



X07796
alpha-amylase


Bacillus subtilis 2633



AY594351
alpha-amylase


Bacillus subtilis strain HA401



AY376455
amy


Bacillus subtilis



V00101
amyE


Bacillus subtilis



AF115340
maltogenic amylase
EC 3.2.1.133

Bacillus subtilis



AF116581
amy
EC 3.2.1.1

Bacillus subtilis



K00563
alpha-amylase


Bacillus subtilis



M79444
alpha-amylase


Bacillus subtilis



DQ852663
alpha-amylase


Geobacillus stearothermophilus



E01181
alpha-amylase


Geobacillus stearothermophilus



E01180
alpha-amylase


Geobacillus stearothermophilus



E01157
alpha-amylase


Geobacillus (Bacillus)







stearothermophilus



Y17557
maltohexaose-producing
3.2.1.133

Bacillus stearothermophilus




alpha-amylase


AF032864
ami
EC 3.2.1.1

Bacillus stearothermophilus



U50744
maltogenic amylase BSMA
3.2.1.133

Bacillus stearothermophilus



M13255
amyS
EC 3.2.1.1

B. stearothermophilus



M11450
alpha-amylase


B. stearothermophilus



M57457
alpha amylase


B. stearothermophilus



X59476
alpha-amylase


B. stearothermophilus



X02769
alpha-amylase


Bacillus stearothermophilus



X67133
BLMA


Bacillus licheniformis



BD249243
Alpha-amylase


Bacillus licheniformis



DQ517496
alpha-amylase


Bacillus licheniformis strain RH






101


DD238309
alpha-amylase


Bacillus licheniformis



E12201
ACID-


Bacillus licheniformis




RESISTANT/THERMOSTABLE



ALPHA-AMYLASE GENE


BD460878
Alpha-amylase


Bacillus licheniformis



DQ407266
amyl thermotolerant


Bacillus licheniformis



AF438149
amy hyperthermostable


Bacillus licheniformis



A27772
amyl thermotolerant
EC 3.2.1.1

Bacillus licheniformis



A17930
Alpha amylase


Bacillus licheniformis



M13256
amyS


B. licheniformis



M38570
alpha-amylase


B. licheniformis



AF442961
alpha-amylase amyA


Halothermothrix orenii



AY220756
alpha amylase


Xanthomonas campestris



AY165038
alpha-amylase


Xanthomonas campestris pv.







campestris



AF482991
alpha-amylase


Xanthomonas campestris pv.







campestris str. 8004



M85252
amy


Xanthomonas campestris



AY240946
amyB


Bifidobacterium adolescentis



EU352611
alpha-amylase


Streptomyces lividans



EU414483
amylase-like gene


Microbispora sp. V2



EU352611
alpha-amylase (amyA)


Streptomyces lividans



D13178
alpha-amylase


Thermoactinomyces vulgaris



EU159580
amylase gene


Bacillus sp. YX



D90112
raw-starch-digesting


Bacillus sp. B1018




amylase


AB274918
amyI, thermostable amylase


Bacillus halodurans



EU029997
Alpha-amylase


Bacillus sp. WHO



AM409179
maltogenic amylase
3.2.1.133

Bacillus sp. US149



AB178478
Alpha-amylase


Bacillus sp. KR-8104



AB029554
alpha-amylase,
3.2.1.1, 3.2.1.3

Thermoactinomyces vulgaris




glucoamylase


DQ341118
alpha amylase
3.2.1.1

Bifidobacterium thermophilum






strain JCM7027


D12818
glucoamylase
3.2.1.3

Clostridium sp.



AB115912
glucoamylase


Clostridium







thermoamylolyticum



AB047926
glucoamylase


Thermoactinomyces vulgaris R-47



AF071548
glucoamylase


Thermoanaerobacterium







thermosaccharolyticum



DQ104609
glucoamylase (gla)


Chaetomium thermophilum



AB083161
glucoamylase


Aspergillus awamori GA I



EF545003
glucoamylase (gla)


Thermomyces lanuginosus



XM_743288
glucan 1,4-alpha-


Aspergillus fumigatus




glucosidase


DQ268532
glucoamylase A


Rhizopus oryzae



DQ219822
glucoamylase b


Rhizopus oryzae



D10460
glucoamylase


Aspergillus shirousami GLA



AJ304803
glucoamylase


Talaromyces emersonii



Z46901
glucoamylase


Arxula adeninivorans



XM_001215158
glucoamylase


Aspergillus terreus NIH2624



AY948384
glucoamylase


Thermomyces lanuginosus



X00712
Glucoamylase
3.2.1.3

A. niger



AB239766
glucoamylase


Fomitopsis palustris



E15692
Glucoamylase


Aspergillus oryzae



E01247
glucoamylase


Rhizopus oryzae



E01175
glucoamylase


Saccharomycopsis fibuligera



E00315
glucoamylase


Aspergillus awamori



DQ211971
gluB


Aspergillus oryzae



AJ890458
gla66


Trichoderma harzianum



X58117
glucoamylase


Saccharomycopsis fibuligera



X67708
1,4-alpha-D-glucan


Amorphotheca resinae




glucohydrolase.


X00548
glucoamylase G1 cDNA


Aspergillus niger



AJ311587
glucoamylase (glu 0111


Saccharomycopsis fibuligera




gene)


AY652617
gluA-A


Aspergillus niger strain






VanTieghem


AY642120
gluA-G


Aspergillus ficuum



AB091510
glucoamylase


Penicillium chrysogenum



AY250996
glucoamylase


Aspergillus niger



AB007825
glucoamylase


Aspergillus oryzae



BD087401
Thermostable glucoamylase


Talaromyces emersonii



BD087377
Thermostable glucoamylase


Aspergillus niger



D00427
glucoamylase I


Aspergillus kawachii



AF082188
GCA1


Candida albicans



AF220541
glucoamylase


Lentinula edodes



D45356
aglA


Aspergillus niger



D00049
glucoamylase


Rhizopus oryzae



D49448
glucoamylase G2


Corticium rolfsii



U59303
glucoamylase


Aspergillus awamori



X13857
Glucan-1.4-alpha-


Saccharomyces cerevisiae




glucosidase


L15383
glucoamylase


Aspergillus terreus



M60207
glucoamylase (GAM1)


Debaryomyces occidentalis



M89475
gla1


Humicola grisea thermoidea



M90490
1,4-alpha-D-


Saccharomyces diastaticus




glucanglucohydrolase


D10461
AMY
3.2.1.1

Aspergillus shirousami



DQ663472
alpha-amylase


Fusicoccum sp. BCC4124



EU014874
AMY1


Cryptococcus flavus



AB083162
amyl III


Aspergillus awamori



AB083160
amyl III


Aspergillus awamori



AB083159
amyl I


Aspergillus awamori



XM_001544485
alpha-amylase A


Ajellomyces capsulatus



EF143986
alpha-amylase


Phanerochaete chrysosporium



EF682066
alpha-amylase


Paracoccidioides brasiliensis



XM_750586
alpha-amylase
3.2.1.1

Aspergillus fumigatus Af293



XM_744365
alpha-amylase AmyA


Aspergillus fumigatus Af293



XM_742412
Maltase
3.2.1.3

Aspergillus fumigatus Af293



XM_001395712
amyA/amyB
3.2.1.1

Aspergillus niger



XM_001394298
acid alpha-amylase


Aspergillus nomius strain PT4



DQ467933
amyl


Aspergillus nomius strain KS13



DQ467931
amyl


Aspergillus nomius strain TK32



DQ467931
amyl


Aspergillus nomius



DQ467923
amyl


Aspergillus pseudotamarii



DQ467918
alpha amylase


Aspergillus parasiticus



DQ467917
amyl


Aspergillus sp. BN8



DQ467916
amyl


Aspergillus flavus strain UR3



DQ467908
amyl


Aspergillus flavus strain AF70



XM_001275450
alpha-amylase


Aspergillus clavatus NRRL



XM_001275449
alpha-glucosidase/alpha-


Aspergillus clavatus




amylase


XM_001265627
alpha-amylase


Neosartorya fischeri NRR




maltase
3.2.1.3

Neosartorya fischeri



DQ526426
amy1
3.2.1.1

Ophiostoma floccosum



EF067865
AMY1


Ajellomyces capsulatus



X12727
alpha-amylase


Aspergillus oryzae



AY155463
alpha-amylase


Lipomyces starkeyi



XM_567873
Alpha-amylase


Cryptococcus neoformans var.







neoformans



BD312604
Alpha-amylase


Aspergillus oryzae



XM_714334
maltase
3.2.1.3

Candida albicans



X16040
amy1
3.2.1.1

Schwanniomyces occidentalis



AB024615
amyR


Emericella nidulans



U30376
alpha-amylase


Lipomyces kononenkoae subsp.







spencermartinsiae



AB008370
acid-stable alpha-amylase


Aspergillus kawachii



K02465
glucoamylase
3.2.1.1

A. awamori



XM_001276751
Maltogenic alpha-amylase
3.2.1.133

Aspergillus clavatus NRRL 1



XM_001273477
Maltogenic alpha-amylase


Aspergillus clavatus NRRL 1



AB044389
Maltogenic alpha-amylase


Aspergillus oryzae



EU368579
Maltogenic alpha-amylase


Bacillus sp. ZW2531-1



M36539
Maltogenic alpha-amylase


Geobacillus stearothermophilus



AM409179
Maltogenic alpha-amylase


Bacillus sp. US149



Z22520
maltogenic amylase


B. acidopullulyticus



X67133
maltogenic amylase


Bacillus licheniformis



AY986797
maltogenic amylase


Bacillus sp. WPD616



U50744
maltogenic amylase


Bacillus stearothermophilus



M36539
maltogenic amylase


B. stearothermophilus



AF115340
maltogenic amylase
3.2.1.133

Bacillus subtilis Bbma



AF060204
maltogenic amylase


Thermus sp. IM6501



Z22520
maltogenic amylase


Bacillus acidopullulyticus



AAF23874
maltogenic amylase


Bacillus subtilis SUH4-2



AY684812
maltogenic amylase


Bacillus thermoalkalophilus






ET2


U50744
maltogenic amylase


Geobacillus stearothermophilus






ET1



LACCASE
1.10.3.2


EU375894
laccase
1.10.3.2

Hypsizygus marmoreus



AM773999
laccase


Pleurotus eryngii



EF175934
laccase (lcc15)


Coprinopsis cinerea strain






FA2222


EU031524
laccase


Pleurotus eryngii var. ferulae



EU031520
laccase


Pleurotus eryngii var. ferulae



EF050079
laccase 2


Sclerotinia minor



AM176898
lac gene


Crinipellis sp. RCK-1



EF624350
laccase


Pholiota nameko strain Ph-5(3)



AB212734
laccase4


Trametes versicolor



Y18012
laccase


Trametes versicolor



D84235
laccase


Coriolus versicolor



AB200322
laccase


Thermus thermophilus



AY228142
alkaline laccase (lbh1)


Bacillus halodurans

















TABLE VI







EC 3.1.1.74










No.
Source
Gene
Accession No.













1

Colletotrichum gloeosporioides

cutinase
M21443


2

Fusarium oxysporum

cutinase (lip1) gene
EF613272


3

Neosartorya fischeri NRRL 181

cutinase family protein
XM_001266631




(NFIA_102190)


4

Neosartorya fischeri NRRL 181

cutinase family protein
XM_001260899




(NFIA_089600)


5

Pyrenopeziza brassicae

cutinase gene
AJ009953


6

Botryotinia fuckeliana

cutA gene
Z69264


7

Ascochyta rabiei

cut gene for cutinase
X65628


8

Aspergillus terreus NIH2624

cutinase precursor
XM_001213969




(ATEG_04791)


9

Aspergillus terreus NIH2624

acetylxylan esterase
XM_001213887




precursor (ATEG_04709)


10

Aspergillus terreus NIH2624

acetylxylan esterase
XM_001213234




precursor (ATEG_04056)


11

Aspergillus terreus NIH2624

cutinase precursor
XM_001212818




(ATEG_03640)


12

Aspergillus terreus NIH2624

cutinase precursor
XM_001211311




(ATEG_02133)


13

Emericella nidulans

cutinase (AN7541-2)
DQ490511


14

Emericella nidulans

cutinase (AN7180-2)
DQ490506


15

Monilinia fructicola

cutinase (cut1) gene
DQ173196


16

Nectria haematococca

cutinase 3 (cut3) gene
AF417005


17

Nectria haematococca

cutinase 2 (cut2)
AF417004


18

Nectria ipomoeae

cutinase (cutA)
U63335


19

Phytophthora infestans clone

cutinase
AY961421



PH026H6


20

Phytophthora infestans

cutinase (Cut1)
AY954247


21

Phytophthora brassicae

cutinase (CutB)
AY244553


22

Phytophthora brassicae

cutinase (CutA)
AY244552


23

Phytophthora capsici

cutinase
X89452


24

Monilinia fructicola

cutinase (cut1)
AF305598


25

Blumeria graminis

cutinase (cut1)
AF326784


26

Glomerella cingulata

cutinase
AF444194


27

Mycobacterium avium

serine esterase cutinase
AF139058


28

Aspergillus oryzae

CutL gene for cutinase
D38311


29

Fusarium solani

cutinase
M29759


30

Fusarium solani pisi

cutinase
K02640


31

Colletotrichum capsici

cutinase
M18033


32

Alternaria brassicicola

cutinase (cutab1)
U03393



















TABLE VII





No.
Source
Gene
Accession No


















1

Colletotrichum gloeosporioides

pectate lyase (pelA) partial
L41646


2

Colletotrichum gloeosporioides

pectin lyase (pnlA)
L22857


3

Bacillus subtilis

pectin lyase
D83791


4

Aspergillus fumigatus

pectin lyase B
XM_743914


5

Aspergillus fumigatus

pectin lyase
XM_748531


6

Aspergillus niger

pectin lyase pelD
XM_001402486


7

Aspergillus niger

pectin lyase pelA
XM_001401024


8

Aspergillus niger

pectin lyase pelB
XM_001389889


9

Aspergillus oryzae

pectin lyase 1 precursor
EF452419




(pel1) partial


10

Pseudoalteromonas haloplanktis

pectin
AF278706




methylesterase/pectate




lyase (pelA)


11

Penicillium griseoroseum

pectin lyase (plg2)
AF502280


12

Penicillium griseoroseum

pectin lyase (plg1)
AF502279


13

Aspergillus niger

rglA gene for
AJ489944




rhamnogalacturonan lyase A


14

Aspergillus niger

pelF gene for pectine lyase
AJ489943




F,


15

Aspergillus niger

plyA gene for pectate
AJ276331




lyase A


16

Mycosphaerella pinodes

pelA
X87580


17

Artificial pelC gene
A12250


18

Artificial pelB gene
A12248


19

Aspergillus niger

pelB gene for pectin lyase B
X65552


20

Aspergillus niger

pelA gene for pectin lyase
X60724


21

Emericella nidulans

pectin lyase
DQ490480


22

Emericella nidulans

pectin lyase
DQ490478


23

Erwinia chrysanthemi

kdgF, kduI, kduD, pelW
X62073




genes


24

Erwinia sp. BTC105

pectate lyase
DQ486987


25

Erwinia chrysanthemi

pelI gene
Y13340


26

Erwinia carotovora

pel1, pel2 and pel3 genes
X81847


27

Bacillus sp.

pelA gene
AJ237980


28

Erwinia chrysanthemi

pelC
AJ132325


29

Erwinia chrysanthemi

pelD
AJ132101


30

Bacillus halodurans strain ATCC

pectate lyase
AY836613



27557


31
Uncultured bacterium clone
pectate lyase gene
AY836652



BD12273


32
Uncultured bacterium clone
Pectate lyase
AY836651



BD9113


33
Uncultured bacterium clone
Pectate lyase
AY836650



BD9318


34
Uncultured bacterium clone
Pectate lyase
AY836649



BD8802


35
Uncultured bacterium clone
Pectate lyase
AY836648



BD9207


36
Uncultured bacterium clone
Pectate lyase
AY836647



BD9208


37
Uncultured bacterium clone
Pectate lyase
AY836646



BD9209


38
Uncultured bacterium clone
Pectate lyase
AY836645



BD7597


39
Uncultured bacterium clone
Pectate lyase
AY836644



BD9561


40
Uncultured bacterium clone
Pectate lyase
AY836643



BD8806


41
Uncultured bacterium clone
Pectate lyase
AY836642



BD9837


42
Uncultured bacterium clone
Pectate lyase
AY836641



BD7566


43
Uncultured bacterium clone
Pectate lyase
AY836640



BD7563


44
Uncultured bacterium clone
Pectate lyase
AY836639



BD9170


45
Uncultured bacterium clone
Pectate lyase
AY836638



BD8765


46
Uncultured bacterium clone
Pectate lyase
AY836637



BD7651


47
Uncultured bacterium clone
Pectate lyase
AY836636



BD7842


48
Uncultured bacterium clone
Pectate lyase
AY836635



BD7564


49
Uncultured bacterium clone
Pectate lyase
AY836634



BD7567


50
Uncultured bacterium clone
Pectate lyase
AY836633



BD8804


51
Uncultured bacterium clone
Pectate lyase
AY836632



BD8113


52
Uncultured bacterium clone
Pectate lyase
AY836631



BD8803


53
Uncultured bacterium clones
Pectate lyase
AY836611 to





AY836630


54

Aspergillus niger

pelA
X55784


55

Aspergillus niger

pelC
AY839647


56

Penicillium expansum

pectin lyase (ple1)
AY545054


57

Blumeria graminis f. sp. tritici

pectin lyase 2-like gene,
AY297036




partial sequence


58

Aspergillus oryzae

pel2 gene for pecyin lyase 2
AB029323


59

Aspergillus oryzae

pel1 gene for pecyin lyase 1
AB029322


60

Colletotrichum gloeosporioides

pectin lyase (pnl1)
AF158256



f. sp. malvae


61

Colletotrichum gloeosporioides

pectin lyase 2 (pnl-2)
AF156984



f. sp. malvae


62

Bacillus sp. P-358

pelP358 gene for pectate
AB062880




lyase P358


63

Aspergillus niger

pectin lyase D (pelD)
M55657


64

Erwinia carotovora

pectin lyase (pnl)
M65057


65

Erwinia carotovora

pectin lyase (pnlA)
M59909


66

Bacillus sp. YA-14

pelK gene for pectate
D26349




lyase


67

Streptomyces thermocarboxydus

pl2 gene for pectate lyase
AB375312


68


69

Pseudomonas viridiflava strain

pectate lyase
DQ004278



RMX3.1b


70

Pseudomonas viridiflava strain

pectate lyase
DQ004277



RMX23.1a


71

Pseudomonas viridiflava strain

pectate lyase
DQ004276



PNA3.3a


72

Pseudomonas viridiflava strain

pectate lyase
DQ004275



LP23.1a


73

Aspergillus fumigatus Af293

pectate lyase A
XM_744120


74

Aspergillus niger CBS 513.88

pectate lyase plyA
XM_0014402441


75

Emericella nidulans

pelA
EF452421


76

Bacillus sp. P-4-N

pel-4B gene for pectate
AB042100




lyase Pel-4B


77

Bacillus sp. P-4-N

pel4A gene for pectate
AB041769




lyase Pel-4A


78

Fusarium solani

pelB
U13051


79

Fusarium solani

pelC
U13049


80

Fusarium solani

pelD
U13050


81

Fusarium solani pisi (Nectria

pectate lyase (pelA)
M94692




hematococca)









Claims
  • 1. A method of degrading a plant biomass sample so as to release fermentable sugars therein, the method comprising obtaining a plant degrading cocktail comprising at least two cell extracts, each cell extract comprising an active plant degrading compound recombinantly expressed in cells from which each said cell extract is derived, said at least two cell extracts being plant extracts or bacterial extracts, or a combination of both; and admixing said plant degrading cocktail with said biomass sample.
  • 2. The method of claim 1, wherein said at least two cell extracts comprise at least one plant cell extract comprising a first plant degrading compound and at least one bacterial cell extract comprising a second plant degrading compound.
  • 3. The method of claim 1, wherein said plant degrading cocktail comprises at least three cell extracts, each of said at least three cell extracts comprising cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, maltogenic alpha-amylase, pectolyase or expansin.
  • 4. The method of claim 1, wherein said plant biomass sample comprises grain and/or grain residues, sugar beet, sugar cane, grasses, wood-based biomass, fruits and/or fruit waste residues, or a combination thereof.
  • 5. The method of claim 4, wherein said plant biomass is corn, wheat, barley, or citrus, or waste residues obtained therefrom.
  • 6. The method of claim 4, wherein said plant biomass is switchgrass.
  • 7. The method of claim 4, wherein said plant biomass is sawdust or otherwise processed wood.
  • 8. The method of claim 4, wherein said plant biomass is sugar cane.
  • 9. The method of claim 4, wherein said plant biomass is citrus peel.
  • 10. The method of claim 4, wherein said plant biomass is sugar beet.
  • 11. The method of claim 1, wherein said plant degrading cocktail further comprises rubisco.
  • 12. A method of producing a plant biomass degrading material comprising producing a first plant comprising chloroplasts that express a first plant degrading enzyme and a second plant comprising chloroplasts that express a second plant degrading enzyme;harvesting said first and second plants; andprocessing said first and second plants to produce an enzyme source comprising a combination of enzymes suitable for mixing with and degrading a biomass sample.
  • 13. The method of claim 12, wherein said first and/or second plant is tobacco.
  • 14. The method of claim 12 wherein said processing comprises homogenizing said first and/or second plant or a portion thereof.
  • 15. The method of claim 12, wherein said producing step comprises at least two of the following: producing a first plant comprising chloroplasts that express cellulase, producing a second plant comprising chloroplasts that express lignanse, producing a third plant comprising chloroplasts that express beta-glucosidase; producing a fourth plant comprising chloroplasts that express hemicellulase; producing a fifth plant comprising chloroplasts that express xylanase; producing a sixth plant comprising chloroplasts that express alpha-amylase; producing a seventh plant comprising chloroplasts that express amyloglucosidase; producing an eighth plant comprising chloroplasts that express pectate lyase; producing a ninth plant comprising chloroplasts that express cutinase; producing a tenth plant comprising chloroplasts that express lipase; producing an eleventh plant comprising chloroplasts that express maltogenic alpha amylase, producing a twelfth plant comprising chloroplasts that express pectolyase or a thirteenth plant comprising chloroplasts that express expansin (e.g. swollenin).
  • 16-19. (canceled)
  • 20. A plant material useful for degrading a plant biomass, said material comprising a first chloroplast genome or genome segment having a heterologous gene that encodes a first plant degrading enzyme and a second chloroplast genome or genome segment having a heterologous gene that encodes a second plant degrading enzyme.
  • 21. The plant material of claim 20 wherein said plant material comprises at least two of the following: a first chloroplast genome or genome segment having a heterologous gene that encodes cellulase, a second chloroplast genome or genome segment having a heterologous gene that encodes lignanase, a third chloroplast genome or genome segment having a heterologous gene that encodes beta-glucosidase; a fourth chloroplast genome or genome segment having a heterologous gene that encodes hemicellulase; a fifth chloroplast genome or genome segment having a heterologous gene that encodes xylanase; a sixth chloroplast genome or genome segment having a heterologous gene that encodes alpha-amylase; a seventh chloroplast genome or genome segment having a heterologous gene that encodes amyloglucosidase; an eighth chloroplast genome or genome segment having a heterologous gene that encodes pectate lyase; a ninth plant chloroplast genome or genome segment having a heterologous gene that encodes cutinase; a tenth chloroplast genome or genome segment having a heterologous gene that encodes lipase; an eleventh chloroplast genome or genome segment having a heterologous gene that encodes pectolyase; a twelfth chloroplast genome or genome segment having a heterologous gene that encodes maltogenic alpha-amylase; or a thirteenth chloroplast genome or genome segment have a heterologous gene that encodes expansin.
  • 22. A plant comprising a plant cell having recombinantly expressing cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase pectolyase, maltogenic alpha-amylase, or expansin, said plant a commercial cultivar of tobacco.
  • 23. The plant of claim 22, wherein said enzyme is in a chloroplast in said cell.
  • 24. The method of claim 1 optimized for digesting a citrus biomass sample wherein said plant degrading cocktail comprises cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase or pectate lyase, or a combination thereof; and said biomass sample is a citrus biomass sample.
  • 25. The method of claim 12, wherein said first and second plants are monocots, dicots or algae or a combination thereof.
  • 26. A homogenized plant material comprising at least two of the following active proteins: cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase or pectolyase, or a combination thereof; and wherein said plant material comprises cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha amylase, amyloglucosidase, pectate lyase, cutinase, lipase, pectolyase or maltogenic alpha-amylase; said plant material optionally comprising rubisco and/or swollenin.
  • 27. The homogenized plant material of claim 26, where the enzyme comprises more than 0.1 percent of total protein in said powdered plant material.
  • 28. The homogenized plant material of claim 26, wherein said plant material comprises rubisco.
  • 29. The homogenized plant material of claim 26 wherein said plant material comprises cellulase, beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase, swollenin or pectate lyase, or a combination thereof expressed in a plant, optionally with an amount of rubisco.
  • 30-48. (canceled)
  • 49. The method of claim 1, wherein said plant material is in powdered form.
  • 50. The method of claim 12, wherein said first or second plant is homoplasmic or heteroplasmic with respect to chloroplasts expressing first or second enzyme, respectively.
  • 51-73. (canceled)
  • 74. The method of claim 1, wherein said plant degrading cocktail includes at least two of the following: beta-glucosidase, xylanase, alpha amylase, amyloglucosidase, pectin lyase or pectate lyase
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. Provisional Patent Application No. 61/032,536 filed Feb. 29, 2008 to which priority is claimed under 35 USC 119 and is incorporated by reference.

Provisional Applications (1)
Number Date Country
61032536 Feb 2008 US