Patent Application
20110059503
1. Field of the Invention
The present invention is generally related to the enantioselective production of amino acids using variant biocatalysts. More particularly, the present invention relates to genetically mutated variants of wild type amino acid oxidase enzymes exhibiting increased activity towards specific amino acid substrates, to produce enantiomerically pure amino acids.
2. Description of the Prior Art
Amino acids and amines in high enantiomeric purity (e.g. >99% enantiomeric excess) are of increasing industrial importance because of their applications as resolving agents, chiral auxiliaries/chiral bases and catalysts for asymmetric synthesis, particularly for pharmaceutical, agrochemical and fine chemical products. Additionally, chiral amino acids and amines possess distinct biological activity, and are therefore in demand as intermediates in the pharmaceutical and agrochemical industries. Non-proteinogenic amino acids and amines are one of the most valuable and rapidly growing classes of chemical compounds used in pharmaceutical, chemical and agrochemical discovery and development. The high value of these compounds is partly due to the difficulty of manufacturing them on a large scale. Part of this difficulty arises because many valuable amino acids and amines exist in two distinct 3-dimensional forms in a mixture that is difficult to separate, and only one form is required for a particular application.
Generally, but with the exception of glycine, each of the common amino acids has a chiral, or asymmetric, α-carbon since there are four different functional groups bonded to the α-carbon. Thus, amino acids can exist as stereoisomers, which are compounds with the same molecular formula but differ in arrangement or configuration of their atoms in space. Enantiomers are two stereoisomers, which are non-superimposable mirror images that can exist for each chiral amino acid. The mirror image pairs of amino acids are designated
Chirality is critical to the function of compounds. In many drug applications, the FDA has mandated that only one enantiomer of a compound may be used in a particular drug, and the opposite enantiomer may not be present at all. Thus, chemical and physical methods have been sought to prepare or separate the individual enantiomers of an amino acid or amine. To meet the industrial demand for these compounds, many methods have been described and developed to prepare enantiomerically pure amino acids and amines. These methods include the physical separation or resolution of enantiomeric pairs using chromatographic or crystallization methods, biocatalytic resolution of enantiomers using enzymes, asymmetric synthesis of single enantiomers using chemo- or biological catalysts, and fermentation methods using engineered microbes. Although each of these approaches has noted advantages in specific instances, each has been limited by narrow applicability to a few families of amino acids required by the industry, and many are inherently compromised by low efficiency and relatively low yields. For example, fermentation methods are limited to the production of natural amino acids, whereas most of the amino acids required for pharmaceutical and agrochemical applications do not occur in nature and accordingly are unsuited for the complex biochemical pathways that are used in fermentative methods of production. Another approach has been to chemically manufacture amino acids and amines as racemic mixtures containing both
As a result of these limitations, more general and robust processes have been sought for the commercial preparation of amino acids, including methods in which a chemical catalyst and a biocatalyst can be used under conditions that permit each catalyst to operate efficiently. In one such example, a process of chemo-enzymatic deracemization has been described in which a racemic mixture of a given amino acid can be converted to an optically pure single amino acid enantiomer with a theoretical yield of 100%, by a method of stereo-inversion. In this approach, a highly enantioselective oxidase biocatalyst is used to generate an imine or a keto acid intermediate from only one enantiomer of a target compound in the mixture. Amino acid oxidases and amino acid deaminases are classes of enzymes that catalyze the enantioselective oxidation of an amino acid to produce the corresponding keto acid. Enzymes that only oxidize
This deracemization method has been shown previously to be applicable to natural amino acids, as well as to some unnatural amino acids. One of the drawbacks of the deracemization methods known in the art is that the natural, or wild type,
The present invention provides a biocatalytic transformation process that is applicable to a diverse range of compounds, by providing a general method to develop amino acids at high optical purity. The present invention uses directed evolution to alter the substrate specificity of a wild type amino acid oxidase and significantly improve its biocatalytic activity towards amino acid substrates of choice, including sterically hindered or bulky amino acids, such as but not limited to, branched chain amino acids, halogenated amino acids, straight chain amino acids, branched alkyl groups, adamantyl groups, and functionalized amino acids. More particularly, the present invention discloses variant amino acid oxidases with increased activity towards amino acids such as but not limited to,
Directed evolution is an iterative procedure whereby random mutagenesis of a DNA sequence encoding a protein (e.g. an enzyme) is combined with a screening or selection regime, selecting variants of the enzyme with enhanced desirable qualities such as, but not limited to, activity towards a given substrate or stability. The present invention involves the mutation of the gene that encodes a
The present invention is directed to mutations in native, or wild type, genes encoding
A first aspect of the present invention is to provide a genetically mutated variant
A second aspect of the present invention is to provide a variant biocatalyst having a genetic mutation comprising at least one amino acid substitution in a region from Gly41 to Asp56 in the wild type
A third aspect of the present invention is to provide a variant biocatalyst having a genetic mutation comprising at least one amino acid substitution in a region from Ile86 to Val118 in the wild type
A further aspect of the present invention is to provide a variant biocatalyst having a genetic mutation comprising at least one amino acid substitution in a region from Ser228 to Met245 in the wild type
Another aspect of the present invention is to provide a biocatalyst having a genetic mutation comprising at least one amino acid substitution in a region from Pro43 to Gln68 in the wild type
A further aspect of the present invention is to provide a biocatalyst having a genetic mutation comprising at least one amino acid substitution in a region from Leu93 to Leu113 in the wild type
The present invention is further directed to a method for preparing enantiopure amino acids using a variant biocatalyst of a wild type enzyme. The variant biocatalyst exhibits increased biocatalytic activity towards an amino acid substrate compared to the wild type enzyme. The method comprises providing a racemic solution of amino acid substrate starting material and contacting the racemic solution with a variant biocatalyst. The method further provides enantioselectively oxidizing a group attached to a chiral center of one enantiomer of the amino acid starting material with the variant biocatalyst to produce an imino acid or keto acid. The imino acid or keto acid is reduced, either simultaneously or sequentially, in the presence of a non-selective reductant to yield a composition comprising both enantiomers of the amino acid substrates. The non-selective reductant may be a transition metal catalyst such as, but not limited to, palladium, platinum, rhodium, ruthenium or iridium supported on platinum, alumina or other supports. Repeated cycles of this process results in a solution comprising only the enantiomer of the amino acid that is not a substrate for the oxidase biocatalyst. In one aspect of the present invention, a solution with only
These and other aspects of the present invention will become apparent to those skilled in the art after a reading of the following description of the preferred embodiment when considered with the drawings, as they support the claimed invention.
The present invention is capable of embodiments in many different forms. Preferred embodiments of the invention are disclosed with the understanding that the present disclosure is to be considered as exemplifications of the principles of the invention and are not intended to limit the broad aspects of the invention to the embodiments illustrated.
The combined use of an enantioselective amino acid oxidase biocatalyst and a non-selective reductant may be used to stereoinvert one enantiomer in a mixture of both enantiomers of an amino acid or amine as shown in
Amino acid oxidase biocatalysts with high enantioselectivity for either
It has been discovered that one of the major limitations of using amino acid oxidases from natural sources for amino acid stereoinversion or amino acid deracemization lies in the range of amino acid substrates that can react with the wild type oxidase biocatalysts at an industrially acceptable level. In many cases, the wild type oxidases show little or no activity towards many valuable amino acids required by industry in applications such as, but not limited to, pharmaceuticals or agrochemicals. Thus, according to the present invention, methods of mutagenesis have been applied to alter the substrate specificity of amino acid oxidases. Assays or screening methods are used to detect oxidase variants, arising by random and/or site-directed mutation that display biocatalytic activity towards substrates of interest.
In one embodiment of the present invention, a random mutagenesis approach is applied using error prone PCR to create large libraries of about 103-106 independent variant
In one embodiment of the present invention, a number of active site positions are disclosed in the DNA sequence of the wild type oxidase enzyme which, when mutated, result in oxidase variants with increased biocatalytic activity towards
An amino acid substitution is the replacement or exchange of the original amino acid in a wild-type or parental protein sequence by another amino acid to produce a variant enzyme. The variant enzyme therefore has an altered protein sequence at the position of the substitution. The present invention identifies amino acid ranges in the wild-type or parental enzyme displaying increased biocatalytic activity towards
When the position of a mutation that influences
The present invention contemplates use of other
In one embodiment of the present invention, the native or variant
The
A 15 ml sterile culture tube containing 5 ml of Luria-Bertani (LB) medium (plus 10 mg/ml chloramphenicol; Cm10) is inoculated with a single colony of RCI100 carrying Plasmid pPOT7/ScDAAO and grown overnight at 30° C. and shaking at 250 rpm. A fresh tube containing 5 ml of LB (Cm 10) is inoculated with 0.1 ml of the over night culture and the tube is incubated for 1 hr at 30° C. while shaking at 250 rpm at which time the incubator is set to 40° C. and the culture is incubated for an additional 4 hrs. The OD600 of the culture is measured and the culture is centrifuged at 4,000 rpm in an Eppendorf™ 5801R centrifuge for 15 minutes at 4° C. The resulting cell pellet is re-suspended once in cold 50 mM phosphate buffer and centrifuged at 4,000 rpm in an Eppendorf™ 5801R centrifuge at 4° C. The pellets are lysed using Bugbuster HT Protein Extraction Reagent by Novagen™, USA according to the manufacturer's instructions. Typically, cell pellets are lysed in an amount of reagent to yield the equivalent of an OD600 of 20. The resulting extract is then assayed using the liquid phase oxidase colorimetric assay as described below.
Liquid Phase Assay.
The liquid phase colorimetric assay is performed in a 96 well plate, in which light absorbance is measured at 510 nm, 30° C. using a VERSAmaX plate reader (Molecular Devices) or equivalent plate reader. The assay mixture comprises 190 μl assay solution and 10 μl enzyme solution. The assay solution (for 20 assays) comprises 2 ml 2×4-AAP (4-aminoantipyrine)/TBHBA (2,4,6-tribromo-3-hydroxybenzoic acid) mixture, 40 μl HRP solution (5 mg/ml), 1360 μl H2O, 400 μl
Oxidase Activity is then Calculated, Using the Beer-Lambert Law, for Variant and Parent Enzymes As Follows:
Plate reader initial rate V0 [mOD/min]
Extinction coefficient of colored product, ε[29,400 M−1 cm−1]
Path length of the assay well, 1 [cm]
V02 [μmoles/min/g]=V01/concentration of enzyme in assay [g/L]
V03 [μmoles/min/mg]=V02/1000
V04 [mmoles/hr/g]=V03×60
The native ScDAAO (pPOT7/ScDAAO) was found to have very little biocatalytic activity on
The ScDAAO gene is mutagenized using a GeneMorph II Random Mutagenesis Kit by Stratagene™, USA based on the protocol supplied with the kit. Plasmid pPOT7/ScDAAO is used as the template at four different concentrations (500, 100, 10, and 0.1 ng), and using SEQ ID No. 2 and SEQ ID No. 3 primers. These primers are homologous to flanking regions of the cloned gene and end at the 5′ ATG start and 3′ TTA stop codon respectively. The primary mutagenesis reaction is carried using an Eppendorf Gene Mastercycler PCR machine by EppendorfrM, Germany using the manufacturers protocol (Instruction Manual #200550, program for single-block temperature cyclers).
The DNA from the primary reaction is then amplified in a secondary PCR reaction as follows:
5) to 2)×30 cycles
7) 4° C. hold
The resulting amplified DNA is purified by TAE agarose gel electrophoresis followed by extraction of the DNA from the agarose using a Qiagen Gel Extraction Kit by Qiagen™, USA. The mutagenized epPCR genes are integrated into a plasmid expression vector (pPOT7) using a modified QuikChange (Stratagene™, USA) procedure that incorporates the mutagenized epPCR genes (megaprimers) rather than the short oligonucleotide primers used in standard procedures known in the art. The product of the megaprimer QuikChange reaction is then digested with Dpn I, to remove the methylated vector template, and 2 μA is used to electroporate electro-competent RCI100. The library size is determined by plating dilutions on LB Cm 10 plates followed by incubation overnight at 30° C. Libraries are diluted to 1 ng/μl, and 1 μl is electroporated into RCI100. The electroporation libraries are plated to a cell density of 500-1000 colonies per plate, incubated over night at 25° C. followed by 6 hr at 40° C. to complete colony growth and induce expression of the ScDAAO variants. The plates are then screened using the solid phase oxidase assay.
Solid Phase Colorimetric Assay: The colonies were lifted from the transformation plates on sterile Whatman No. 1, 8.5 cm filter paper circles. Filters are frozen by dipping in liquid nitrogen for 20 seconds (or by freezing at −80° C. for 10 min) and placing on tissue paper with the colony side up to thaw. The solid phase and liquid phase colorimetric assays rely on the detection of the hydrogen peroxide by-product of a successful oxidase reaction. Solid phase assay mixture is prepared as follows:
DAB tablet (Sigma, D-4418)
2 ml 1 M potassium phosphate buffer (pH 7.6)
0.1 ml 5 mg/ml horseradish peroxidase (Sigma, P6782)
2 ml
The master mix is filtered using a 45 μM filter prior to use, then 2.2 ml of the mixture is added a Petri dish containing an 8.5 cm filter paper (Whatman, Grade 1, 1001-085). The filter papers carrying the lifted colonies are overlaid on the filter papers saturated in the assay mixture and the dish is incubated at room temperature for 2 hrs to 72 hrs to detect color. The lifted colonies are observed for up to 72 hrs and positive colonies, which are detected by an increase in brown color relative to non-mutated control, are picked and purified by re-screening again. From this screen four distinct variants with increased activity on
The His141Tyr and Thr218Ile mutations described in Example 1 are combined using the QuikChange procedure by Stratagene™, USA. The His141Tyr mutation is added to the Plasmid pPOT7/ScDAAO C2 variant (Thr218Ile) with a QuikChange II XL kit using the manufacturers recommended conditions, and using mutagenic primers SEQ ID No. 10 and SEQ ID No. 11. Several isolates are sequenced to confirm the correct DNA sequence, as identified in SEQ ID No. 12, and a confirmed clone, Plasmid pPOT7/ScDAAO 9-1, is analyzed for activity against several
Plasmid pPOT9/ScDAAO 9-1 is subject to mutagenesis using the GeneMorph II Random Mutagenesis kit. The mutagenesis, screening, DNA sequencing and specific activity determination were performed as described in Example 2 with one exception. To decrease the background of active colonies, the library colony lifts were heated at 55° C. for 90 minutes prior to activity screening. These conditions completely inactivate the oxidase activity in induced colonies of RCI100 carrying Plasmid pPOT9/ScDAAO 9-1. Thus, any variants having biocatalytic activity after heat treatment should be more active and/or more heat stable and yield a more robust oxidase. As shown in Table 3, a number of improved variants exhibiting higher specific biocatalytic activity on
Asp7Glu
Glu62Lys
Gln68Arg
Val50Ala
Leu93Val
Glu99Gly
Ala211Thr
Trp108Arg
Variant pPOT9/ScDAAO 1-26 (SEQ ID No. 16) is subject to further mutagenesis using the GeneMorph II Random Mutagenesis kit (Stratagene, CA). The mutagenesis, screening, DNA sequencing and specific activity determination are performed as described in example 4 except the screening stringency was increased to 2.5 hr at 60° C. Under these conditions the activity of the enzyme from RCC0069 (pPOT9/ScDAAO 1-26) is completely inactivated. Colonies exhibiting activity after this treatment are assayed as previously described. After DNA sequencing the exact amino acid changes were determined as shown in Table 4. Several of these variants exhibited increased specific activity on
The
Primers are designed, as identified by SEQ ID Nos. 37 and 38 so that Tyr232, equivalent to positions Tyr243 in T. variabilis
The gene encoding T. variabilis
Saturation mutagenesis of the gene encoding T. variabilis
The plasmid borne libraries were used to transform E. coli BW22513 by electroporation and the transformants were plated to a density of 500-1000 colonies per plate, incubated over night at 25° C. followed by 6 hr at 40° C. to complete colony growth and induce the T. variabilis DAAO variants. The plates can then be then screened using a solid phase oxidase assay as described above in example 1.
A number of variants were obtained with significantly enhanced biocatalytic activity on
The variant pPOT3/TvDAAO TBG22 was chosen as the template for further iterative mutagenesis. Sequence alignment of various DAAO protein sequences demonstrates that the equivalent position of His141 of S. coelicolor DAAO, which was shown to improve the activity of this enzyme towards
Further random mutagenesis was performed using pPOT3/TvDAAO TBG22 and TBG26 as the templates. Libraries of randomly mutated variants were produced using the GeneMorph II Random Mutagenesis kit (Stratagene, CA) and screened for increased heat stability as described above.
The mutations Gly41Arg and Ala106Thr were identified as providing increased activity and thermostability using the solid phase plate screen. These mutations were incorporated into the pPOT3/TvDAAO TBG29 template using the Quikchange procedure to produce the variant pPOT3/TvDAAO TBG39. In addition, it is noted that Ala106 of TvDAAO is in alignment with Gly101 of S. coelicolor, which was found herein to offer increased activity when mutated to Ser. This evidence further establishes the way that similar mutations in homologous proteins can be expected to have similar effects, even if the specific oxidase has not been studied previously.
Further screening of the random epPCR libraries yielded a number of variants that were identified with increased activity and/or stability. Three of the most active in the screen (darkest colonies) had the mutations Tyr55Ser, Asp56Glu and Ala103Pro. These mutations were introduced into the variant pPOT3/TvDAAO TBG39 using the QuikChange protocol and are named pPOT3/TvDAAO TBG40, pPOT3/TvDAAO TBG45 and pPOT3/TvDAAO TBG47 for the incorporation of Tyr55Ser, Tyr55Ser/Asp56Glu and Tyr55Ser/Asp56Glu/Ala103Pro respectively. Furthermore, the mutations Asp56Pro and Tyr55Thr were also found to offer similar advantages. Other mutations yielding variants with increased activity and/or stability but were weaker in the screen than those described above include Gly41Ala, Ala51Thr, Asp64Asn, Glu73Lys, Ser77Asn, Cys108Ser, Met156Ile, Asp188Ala, Lys225Asn and Ser228Thr. It would be expected that combinations of these mutations or the corresponding positions in other homologous DAAOs would lead to variants with improved activity, specificity and/or stability.
This variant was named pPOT3/TvDAAO WT1 and was produced using the QuikChange procedure and the pPOT3/TvDAAO TBG39 template to change Met241 and Val243 back to the wild type amino acids (Ala241 and Tyr243). The resulting variant pPOT3/TvDAAO WT1 was tested for improved heat stability as shown in
Detailed structural modeling to further examine the TvDAAO enzyme identified Ser228 as a possible position in TvDAAO for mutagenesis to modulate substrate specificity. The Ser228 position of TvDAAO was subjected to saturation mutagenesis as described above using the gene encoding the enzyme pPOT3/TvDAAO WT1 as the template as pPOT3/TvDAAO WT1 displayed low activity against
The Ser228 position of TvDAAO was subjected to saturation mutagenesis as described above using the gene encoding the enzyme pPOT3/TvDAAO TBG47 as the parent. The library was screened against
Enzyme variants were also assayed against
The results of the TvDAAO mutagenesis demonstrate that incorporating mutations that increase the specificity towards
It is noted that the data presented herein identifies certain trends and specific regions of the protein that enhance the activity and stability of a family of oxidase proteins. As discussed above, mutations of proteins, and substantially similar homologous proteins, in these regions should offer similar benefits and these regions constitute the results of the present invention.
For example, one region is encompassed by the amino acids Gly41 to Asp56 in TvDAAO, which includes the specific amino acids Gly41, Tyr55 and Asp56 that have been mutated in this work and shown to be beneficial. The equivalent region in S. coelicolor DAAO is Pro43 to Gln68 and includes the amino acids Val50, Arg60, Glu62 and Gln68 that have been mutated in this work and shown to be beneficial.
A second region that has been shown to have a strong effect on oxidase stability is encompassed by the amino acids Ile86 to Val118 in TvDAAO, which includes the specific amino acids Ala103 and Ala106 that have been mutated in this work and shown to be beneficial. The equivalent region in S. coelicolor DAAO is Leu93 to Leu113 and includes the amino acids Leu93, Glu99, Thr100, Gly101, Asp106, Trp108, Ala111 and Leu113 that have been mutated in this work and shown to be beneficial.
In general, the mutations and regions described herein are unique, novel and could not be predicted. Additionally, literature reports of mutations in TvDAAO have focused either on stability improvements or biochemical analyses rather than substrate specificity modulation as described herein. Furthermore, the published crystal structures of the pig kidney and R. gracilis enzymes have identified the active site amino acids in these enzymes which naturally suggest important amino acids that could be targeted for mutation. However, these rational approaches often do not produce the expected results and a multi-faceted approach as described herein is necessary.
A culture of recombinant E. coli containing a cloned gene, encoding a suitable amino acid oxidase biocatalyst, such as Trigonopsis variabilis
Preparation of the oxidase biocatalyst. Cells prepared by fermentation are lysed using a French Pressure Cell. The resulting lysate has 0.2% (w/v) polyethyleneimine added, and is centrifuged at 10,000×G for 30 minutes at 4° C. The pellet is discarded and the supernatant containing the oxidase biocatalyst is recovered and stored at −20° C.
Racemic tert-leucine (26.2 g, 0.1998 mole) is dissolved in water (350 ml) and adjusted to pH 7.5 with 1 M sodium hydroxide. The racemic tert-leucine solution is transferred to a temperature controlled jacketed reaction vessel at 30° C. and agitated at 200 rpm using an overhead stirrer. Oxygen gas is sparged into the reaction solution at 0.5 L/min. Micrococcus catalase (1 ml) and 50% polypropylene glycol (1 ml) is added. T. variabilis pPOT3/TvDAAO TBG40 (148 ml cell free extract solution prepared from 22.5 g of cells) is then charged to the vessel and the reaction is monitored by HPLC analysis. Chiral HPLC analysis indicated the reaction reached completion in 19 hr, (
Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing description. The above-mentioned examples are provided to serve the purpose of clarifying the aspects of the invention and it will be apparent to one skilled in the art that they do not serve to limit the scope of the invention. While the specific embodiments have been illustrated and described, numerous modifications come to mind without significantly departing from the spirit of the invention, and the scope of protection is only limited by the scope of the accompanying claims.
This application claims priority to U.S. Provisional Patent Application Ser. No. 60/905,753 filed Mar. 8, 2007, the entire disclosures of which are incorporated herein by reference. Priority to this application is claimed under 35 U.S.C. §§119 and/or 120.
| Number | Date | Country | |
|---|---|---|---|
| 60905753 | Mar 2007 | US |
