Patent Application
20160060608
The present invention relates to novel lipases isolated from the bovine rumen metagenome and from Anaerovibrio lipolytica 5S and polynucleotides encoding the lipases. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the lipases as well as methods of producing and using the lipases.
The rumen is the primary site of lipid hydrolysis and transformation in ruminants, and lipid metabolism plays a significant role in regulating the overall lipid composition of microbial cells and also of milk and meat of the ruminant animal (Harfoot and Hazlewood, 1997; Scollan et al., 2006; Lourengo et al., 2010). The lipid content of forage ingested by ruminants ranges from 2 to 10% of the total dry weight (Harfoot and Hazlewood, 1997), which represent 1.5 kg of ingested lipids through forage per day by dairy cattle (Harfoot, 1978) and nearly 550 kg of lipid per year (Jarvis and Moore, 2010).
Dietary lipids enter the rumen either as triglycerides (neutral lipids) in concentrate-based feeds or as glycolipids or phospholipids (polar lipids) in forages (Harfoot and Hazlewood, 1997; Bauman et al., 2003). Other polar lipids, like sulfolipids, are also present as minor components in forage (<5%) (Harfoot and Hazlewood, 1997). On entering the rumen, lipids are hydrolyzed by lipases, which results in the liberation of glycerol and unsaturated and saturated fatty acids. These fatty acids go through microbial biohydrogenation and are transformed to more saturated end products.
Research on lipid metabolism in the rumen has largely focused on biohydrogenation of polyunsaturated fatty acids with an emphasis on the intermediates formed, in particular conjugated linoleic acids, due to the potential of these molecules to affect human health (Enjalbert and Troegeler-Meynadier, 2009). There is a dearth of data on microbial lipolysis, the first step in lipid metabolism in the rumen. It is known that dietary lipids are predominantly hydrolyzed in the rumen by obligatory anaerobic bacteria (Jenkins et al., 2008) and there is no evidence of rumen protozoa or fungi being significantly involved in ruminal lipolysis (Harfoot and Hazlewood, 1997; Jenkins et al., 2008, Lourengo et al., 2010).
However, to date, only six pure cultures of obligatory anaerobic, lipolytic bacteria have been isolated from the rumen, belonging to the genera Anaerovibrio, Butyrivibrio, Clostridium and Propionibacterium (Jarvis and Moore, 2010), and only a small number of lipolytic enzymes have been retrieved from the rumen metagenome of either cattle (Liu et al., 2009) or sheep (Bayer et al., 2010).
Hobson and Mann (1961) isolated a bacterium from the sheep rumen able to hydrolyze linseed oil triglycerides to glycerol and fatty acids, using anaerobic techniques and a combination of differential and selective media (Stewart et al., 1997). It was named Anaerovibrio lipolytica (Hungate, 1966), or A. lipolyticus (Strompl et al., 1999). The growth characteristics of strain 5S were described in continuous culture. Ribose, fructose and D-lactate were used as growth substrates and glycerol was fermented to propionate, lactate and succinate (Hobson and Summers, 1966, 1967; Henderson et al., 1969). Extracellular lipase activity was characterized in cell free-medium and after purification by chromatography on Sephadex columns; the lipases were most active at pH 7.4 and 20 to 22° C., and diglycerides were hydrolyzed more rapidly than triglycerides (Henderson, 1970, 1971; Henderson and Hodgkiss, 1973). Ruminal lipase activity in animals receiving mainly concentrate feeds is thought to be accomplished mainly by A. lipolytica, although other lipolytic species might be expected to predominate in grazing animals as A. lipolytica lacks the ability to hydrolyze galacto- and phospholipids (Henderson, 1971). These latter lipids are known to be hydrolyzed in vitro by the Butyrivibrio fibrisolvens strains S2 and LM8/1B (Harfoot and Hazlewood, 1997). A. lipolytica was shown to be present at around 107/ml in grazing animals (Prins et al., 1975). Thus it has been hypothesized that A. lipolytica could have other activities, such as the fermentation of glycerol (Rattray and Craig, 2007). Other molecular microbial ecology studies have enumerated A. lipolytica in the rumen under different dietary conditions (Tajima et al., 2001; Koike et al., 2007; Huws et al., 2010) with similar conclusions. These studies have shown that A. lipolytica is predominant and one of the best recognized ruminal lipolytic bacteria. However no recent studies have been undertaken to enhance our knowledge of its lipase/s or genomic features using available modern laboratory techniques.
As lipases are used in a wide variety of applications, such as processing of fats and oils, detergent compositions and diagnostic reagents, there is a need for new and alternative lipolytic enzymes and their uses in biotechnological applications.
It has been found that the bovine rumen metagenome harbours a number of active lipases. As such, these lipases and nucleic acid sequences encoding these lipases can be used in the preparation of polymers, in formulations which may comprise detergents, as catalysts, as part of diagnostic kits, in the preparation of biofuels and for the prevention and/or treatment of a disease.
In one aspect, the present invention relates to a lipase comprising a polypeptide or peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19, or an amino acid sequence that has at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity therewith.
Preferably, the lipase comprising a polypeptide or peptide comprising one of the sequences of SEQ ID NO: 1-5 belongs to lipase family 1.7. The lipase comprising a polypeptide or peptide comprising the sequence of SEQ ID NO: 6 belongs to lipase family II. The lipase comprising a polypeptide or peptide comprising one of the sequences of SEQ ID NO: 7-12 belongs to lipase family VII. The lipase comprising a polypeptide or peptide comprising one of the sequences of SEQ ID NO: 13 and 14 belongs to the HSL lipase family (family IV). The lipase comprising a polypeptide or peptide comprising one of the sequences of SEQ ID NO: 15 and 16 belongs to the lipase family VIII. The lipase comprising a polypeptide or peptide comprising one of the sequences of SEQ ID NO: 17 and 18 belongs to the GDSL family. The lipase comprises a polypeptide or peptide comprising the sequences of SEQ ID NO: 19 belongs to family V.
Preferably, the lipase is an isolated lipase.
According to another aspect of the present invention, there is provided a lipase derived from bovine rumen metagenome.
According to another aspect of the present invention, there is provided a lipase derived from Anaerovibrio lipolytica 5S.
According to another aspect of the present invention, there is provided a nucleic acid sequence which encodes a lipase of the present invention or a fragment or variant thereof.
Preferably, the fragments or variants of the nucleic acid sequence of the present invention comprise a nucleic acid sequence that is hybridisable thereto under stringent conditions and/or a nucleic acid sequence that is complementary thereto.
Accordingly, in one aspect of the present invention, there is provided a nucleic acid sequence which is (i) complementary to a nucleic acid sequence which encodes a peptide or a polypeptide of the present invention; and/or (ii) hybridizable to a nucleic acid sequence which encodes a peptide or polypeptide of the present invention.
Preferably, the nucleic acid sequence is an isolated nucleic acid sequence.
Also provided by the present invention is a nucleic acid molecule comprising a nucleic acid sequence of the present invention.
Preferably, the nucleic acid molecule comprises single or double stranded DNA or RNA.
Preferably, the nucleic acid molecule further comprises vector nucleic acid sequences.
Preferably, the nucleic acid molecule further comprises nucleic acid sequences encoding a heterologous polypeptide.
Another aspect of the present invention relates to an antibody, that binds specifically with one or more lipases of the invention.
Another aspect of the present invention relates to a host cell which contains the nucleic acid molecule of the present invention.
The host cell may be a mammalian host cell or a non-mammalian host cell.
Preferably, the nucleic acid sequence is incorporated into a vector, for example a DNA plasmid. As such, in one aspect of the present invention, there is provided a vector, for example a DNA plasmid, comprising a nucleic acid sequence of the present invention.
Preferably, the plasmid is a fosmid or an open reading frame (ORF) in a plasmid.
Another aspect of the invention is a process for the preparation of a lipase of the present invention, comprising the steps of culturing an organism that expresses the lipase in a culture medium and recovering the lipase from the culture medium.
Preferably, the organism is a host cell. Preferably, the host cell may be a mammalian host cell or a non-mammalian host cell.
It is also provided a lipase obtainable by the process for the preparation of the lipase.
Preferably, the expression takes place in a suitable host cell. The host cell may be transformed with a polynucleotide or polynucleotide fragment selected from the group consisting of SEQ ID NO: 20-38, or a polynucleotide or polynucleotide fragment with sequence that has at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% nucleic acid sequence identity therewith.
Preferably, the lipase obtainable from the expression of a sequence selected from the group of SEQ ID NO: 20-24 belongs to lipase family 1.7. The lipase obtainable from the expression of a sequence of SEQ ID NO: 25 belongs to lipase family II. The lipase obtainable from the expression of a sequence selected from the group of SEQ ID NO: 26-31 belongs to lipase family VII. The lipase obtainable from the expression of a sequence selected from the group of SEQ ID NO: 32-33 belongs to the lipase family IV (HSL lipase family). The lipase obtainable from the expression of a sequence selected from the group of SEQ ID NO: 34-35 belongs to lipase family VIII. The lipase obtainable from the expression of a sequence selected from the group of SEQ ID NO: 35-37 belongs to the GDSL family. The lipase obtainable from the expression of a sequence selected from the group of SEQ ID NO: 38 belongs to family V.
Another aspect of the invention is the lipase of the invention immobilized on a solid carrier material. The carrier material may be a polymer support, a resin, a macroporous resin, celite or silica gel and/or a combination thereof. The polymer support may be a polystyrene support or an acrylic support.
A further aspect of the invention is the use of the lipases of the present invention in the preparation of a polymer. The polymer may be a bioplastic. The polymer may be biocompatible. The polymer may be biodegradable. The polymer may be in the form of a film, a fibre or a nanoparticle. The polymer may be amorphous or crystalline. Preferably, the polymer comprises lactic acid subunits. The polymer may be a copolymer. The copolymer may comprise a diacid, a diol or a hydroxyl acid. The polymer may be a high molecular weight polymer.
Another aspect of the invention is a process comprising reacting one or more substrates in the presence of the lipase of the present invention. The substrate may be selected from the group of a lactide, a cyclic lactide dimmer and a low molecular weight polylactic acid oligomer. The lactide may be selected from the group of an L-lactide, a D-lactide and a DL-lactide. Preferably, the process is selected from the group of polymerization, ring opening polymerization reaction and an enzymatic copolymerization reaction. Preferably, the product of the process is a high molecular weight polymer. The polymer produced by the process may be a copolymer.
According to another aspect of the invention, there is provided a lipase formulation comprising the lipase of the present invention. The formulation may further comprise a detergent. The formulation may comprise, in addition to the lipase, stabilizers, further detergents, enzyme substrates or combinations thereof.
Another aspect of the invention is the use of the lipase of the present invention as a catalyst.
Another aspect of the invention is a process for the enzyme-catalytic conversion or enantioselective conversion of substrates comprising reacting substrates in the presence of the lipase of the present invention. Substrates of the invention may be alcohols, amines, amino acid esters, carboxylic acid esters.
Another aspect of the invention is a process for the preparation of optically active compounds comprising reacting stereoisomeric mixtures or racemates of a substrate in an enzyme catalyzed manner enantioselectively in the presence of a lipase of the present invention, and resolving the mixture.
Another aspect of the invention is a diagnostic kit comprising the lipase of the present invention. Preferably, the kit further comprises an antibody and a probe.
A further aspect of the invention is a diagnostic kit comprising an antibody binding to the lipase of the present invention.
A further aspect of the invention is a method of diagnosis comprising contacting a sample with a lipase or an antibody binding to the lipase of the present invention. The diagnosis may be for the diagnosis of blood lipid values or the presence of virulence factors.
A further aspect of the invention is the use of the lipase of the present invention in the preparation of biofuels. Preferably, the biofuel is biodiesel.
Another aspect of the invention is the use of the lipase of the present invention for oil biodegradation, waste treatment, leather degreasing.
Another aspect of the present invention relates to a peptide or polypeptide of the present invention and/or a nucleic acid sequence and/or an antibody of the present invention for use in therapy.
A further aspect of the present invention relates to a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences and/or two or more antibodies of the present invention for use in therapy.
Another aspect of the present invention relates to use of a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention and/or an antibody of the invention in therapy.
A further aspect of the present invention relates to use of a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention and/or two or more antibodies of the present invention in therapy.
Another aspect of the present invention relates to a method for treating a patient with a disease, the method comprising administering to a patient a therapeutically effective amount of a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention and/or an antibody of the present invention.
Another aspect of the present invention relates to a method for treating a patient with a disease, the method comprising administering to a patient a therapeutically effective amount of a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention and/or a combination of two or more antibodies of the present invention.
A further aspect of the present invention relates to a method for treating a patient, the method comprising administering to a patient a therapeutically effective amount of a peptide or polypeptide of the present invention and/or a nucleic acid sequence and/or an antibody of the present invention.
A further aspect of the present invention relates to a method for treating a patient, the method comprising administering to a patient a therapeutically effective amount of a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention and/or a combination of two or more antibodies of the present invention.
Another aspect of the present invention relates to a composition comprising a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention and/or an antibody of the present invention.
A further aspect of the present invention relates to a composition comprising a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention and/or a combination of two or more antibodies of the present invention.
Preferably, the composition is a pharmaceutical composition.
An embodiment of the present invention will now be described by way of example, with reference to the accompanying figures in which;
It will of course be understood that many variations may be made to the above-described embodiment without departing from the scope of the present invention.
The invention provides lipases and for polynucleotides encoding the lipases.
In another aspect of the invention, the lipase of the present invention is used as a catalyst in a process for the preparation of a polymer. The polymer may be a bioplastic. The polymer may be biodegradable. The polymer may be polylactic acid polymer (PLA). The polymer may be an amorphous or crystalline polymer. The polymer may be moulded into parts, film, fibre and/or nanoparticles.
The PLA polymer may be prepared from a lactide, a cyclic lactide dimer, a low molecular weight polylactic acid oligomer or a lactide in combination with a copolymer. The lactide may be selected from the group of an L-lactide, a D-lactide and DL-lactide. The copolymer may be a diacid, diol or hydroxyl acid. The PLA may be prepared by a process of lipase-catalyzed ring opening polymerization.
A further aspect of the invention is a lipase as mentioned above for use in a detergent composition for use in dish washing, bleaching composition, decomposition of lipid contaminants in dry cleaning solvents, liquid leather cleaner, contact lens cleaning, cleaning of drains clogged by lipids in food processing or domestic/industrial effluent treatment plants, degradation of organic wastes on the surface of exhaust pipes, toilet bowls, removal of dirt/cattle manure from domestic animals by lipases and cellulases, washing, degreasing and water reconditioning by using lipases along with oxidoreductases, which allows for smaller amounts of surfactants and operations at low temperatures.
Another aspect of the invention is the use of a lipase as mentioned above in food processing. For example, a lipase of the invention may be used in the removal of fat from meat, including fish meat. The fat may be removed from the meat by adding a lipase during the processing of the meat. The lipase may be used in the manufacture of sausages and for determining the changes in the long-chain fatty acids liberated during the ripening of the sausages. The lipase may be used for refining rice flavour modifying soybean milk, for improving the aroma and accelerating the fermentation of apple wine. The lipase can be used for the preparation of enzyme modified cheeses. The lipase can be used for the enhancement of flavour of dairy products. Examples of such products are cheese, coffee whiteners, milk, butter, yoghurt. In this regard, the lipase can be used for accelerating the ripening of cheese products.
In another aspect of the invention, the lipase mentioned above is used in a process for the enantioselective enzyme-catalytic conversion of substrates using the lipase. Lipases as mentioned above can be used for the separation of stereoisomers and in particular for the separation of enantiomers or diastereomers from a stereoisomer mixture of the substrate. It can be used for the separation of enantiomers or diastereromers from racemic substrates and thus for the preparation for optically active compounds from the respective racemic mixtures. The resulting products can be easily separated in a manner know per se by chemical, physical and mechanical separation methods. Crystallisation, precipitation, extraction in two-phase solvent systems, chromatographic separation processes such as HPLC, GC or column chromatography on silica gel or thermal separation processes such as distillation are mentioned by way of example.
In a further aspect of the invention, the lipases as mentioned above are used for the preparation of biofuels. The term biofuel includes biodiesel fuel. Biofuels can be generated from vegetable oils.
Definitions
The following definitions shall apply throughout the specification and the appended claims.
Embodiments have been described herein in a concise way. It should be appreciated that features of these embodiments may be variously separated or combined within the invention.
Within this application, the term “lipase” is taken to mean any polypeptide, peptide or enzyme, including carboxyesterases, true lipases and phospholipases, with lipolytic activity.
Within the context of the present application, the term “comprises” is taken to mean “includes among other things”, and is not taken to mean “consists of only”.
Within this specification, the term “about” means plus or minus 20%, more preferably plus or minus 10%, even more preferably plus or minus 5%, most preferably plus or minus 2%.
As used herein, the term “functional fragments or variants thereof” means a fragment or variant of the claimed peptide or polypeptide which has lipolytic activity.
The term “isolated” means substantially separated or purified away from contaminating sequences in the cell or organism in which the nucleic acid or polypeptide naturally occurs and includes nucleic acids and polypeptides purified by standard purification techniques as well as nucleic acids prepared by recombinant technology and nucleic acids and polypeptides and polypeptide fragments chemically synthesised.
“Interact” means affecting the binding or activity of a molecule. This includes competitive binding, agonism and antagonism.
“Pharmaceutically acceptable” means being useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes being useful for veterinary use as well as human pharmaceutical use.
“Treatment” as used herein includes prophylaxis of the named disorder or condition, or amelioration or elimination of the disorder once it has been established.
“An effective amount” refers to an amount of a compound that confers a therapeutic effect on the treated subject. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
The term “antibody” is interpreted to mean whole antibodies and biologically functional fragments thereof. Such biologically functional fragments retain at least one antigen binding function of a corresponding full-length antibody (e.g., specificity for one or more of the peptides or polypeptides of the invention).
Within this specification, “identity,” as it is known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Percentage identity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), all of which are incorporated herein by reference in their entirety. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Preferred computer program methods to determine percentage identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984), which is incorporated herein by reference in its entirety), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990), which is incorporated herein by reference in its entirety). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990), which is incorporated herein by reference in its entirety). As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” to a reference nucleotide sequence of “SEQ ID NO: A” it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of “SEQ ID NO: A.” In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of “SEQ ID NO:B” is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of “SEQ ID NO: B.” In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a receptor at least 50% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 65%, at least about 70%, or at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6. 3.1-6.3.6, which is incorporated herein by reference in its entirety. One example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65° C. In one embodiment, an isolated receptor nucleic acid molecule that hybridizes under stringent conditions to the sequence of SEQ ID NO:1 corresponds to a naturally occurring nucleic acid molecule.
As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e. g., encodes a natural protein).
As used herein, enzyme-catalytic conversions refers to chemical reactions of substrates which are catalyzed by lipases. The following reactions are examples of such reactions, but are not considered as limiting: acylation or enantioselective acylation of alcohols, acylation or enantioselective acylation of amines, acylation or enantioselective acylation of amino esters, such as, for example, amino acid esters, hydrolysis or enantioselective hydrolysis of carboxylic acid esters, ring opening polymerization.
“High molecular weight polymer” means a polymer having a molecular weight above 50,000.
“Low molecular weight polymer” means a polymer having a molecular weight between 1000 and 10000.
It will be understood that the term “biocompatible” means that the polymer is at least non toxic to cells and/or living tissues.
Within this specification embodiments have been described in a way that enables a clear and concise specification to be written, but it will be appreciated that embodiments may be variously combined or separated without parting from the invention.
The invention will now be further illustrated by the following non-limiting examples.
Materials and Strains
All chemicals used for enzymatic tests and library screening were of the purest grade available and were purchased from Sigma-Aldrich Company Ltd. (Dorset, UK), Fisher Scientific (Leicestershire, UK) and TCI Europe (Zwijndrecht, Belgium). Restriction enzymes were from Promega UK Ltd. (Southampton, UK). Escherichia coli strains EPI300-T1R (Epicentre, Cambio Ltd., Cambridge, UK) were used for library construction and screening, and TOP10 (Invitrogen, Carlsbad, Calif., USA), for protein expression. All bacterial hosts were maintained and cultivated according to the supplier's recommendations.
Rumen Sampling and DNA Extraction
Rumen contents were collected from four rumen-fistulated, non-lactating Holstein cows (average weight of 731 kg) housed at Trawsgoed experimental farm (Aberystwyth, Ceredigion, Wales). The animals were fed a diet composed of a mixture of grass silage and straw (75:25) ad libitum and ˜1 kg of sugar beet nuts at 0700 with constant access to fresh water. Sampling was completed 2 h after concentrate feeding. Strained ruminal fluid (SRF), solid-attached bacteria (SAB) and liquid associated bacteria (LAB) were harvested as described by Huws et al. (2010).
Construction of Metagenomic Libraries
Metagenomic DNA was extracted from 200 μl of SRF, SAB and LAB using the BIO101 FastDNA® Spin Kit for Soil (Qbiogene, Cambridge, UK) following the supplier's protocol except that after the first step the samples were shaken three times for 30s, in a FastPrep bead beater (Qbiogene, Cambridge, UK) at 6 m/s with cooling on ice for 30 s between each shake. The libraries were constructed using the CopyControl™ pCC1FOS™ vector and the reagents supplied in the CopyControl™ Fosmid Library Production Kit (Epicentre, Cambio Ltd., Cambridge, UK), following the supplier's recommendations. All clones were picked using a colony picker Genetix QPix2 XT (Genetix Ltd., New Milton, England), and sub-cultured for 20 h in 384-well plates (Genetix Ltd., New Milton, England) containing LB broth with 12.5 μg/ml chloramphenicol and 20% glycerol. They were then stored at −80° C.
Screening for Lipase Activity
Selective screening of the clones for lipolytic activity was accomplished using spirit blue agar (Sigma-Aldrich Ltd., Dorset, UK) supplemented with 1% tributyrin and LB agar with 1% (w/v) trioleoyglycerol and 0.001% (w/v) rhodamine B (Kouker and Jaeger, 1987). The media were supplemented with 12.5 μg/ml chloramphenicol for selection and 2 m1/I of Copy-Control Fosmid Autoinduction solution (Epicentre, Cambio Ltd., Cambridge, UK) for high-copy number induction of the clones. The media was poured into square plates (22×22 cm) and the clones stamped onto the agar using a 384-pin replicator (Genetix Ltd., New Milton, England). After incubation for 48 h at 37oC, clones surrounded by a blue precipitate on spirit blue agar were selected. Positive clones were re-tested for lipase activity with a secondary screening, and their fosmids were extracted using the QlAprep® Spin Miniprep kit (Qiagen, Crawley, UK) following the supplier's recommendations. The fosmid size was determined after restriction with BamHI and analysis on an agarose gel.
The libraries consisted of a total of 23,872 clones: 7,744 from SRF, 8,448 from SAB and 7,680 from LAB, with an average insert size of 30-35 kbp. The libraries were screened using both a spirit blue and a trioleoylglycerol-rhodamine B assay. Five clones from the SAB library and four clones from the LAB library were positive for lipolytic/esterase activity with the spirit blue assay, while no positive clones were identified with the trioleoylglycerol-rhodamine B assay. There were no positive clones observed from the SRF library. The SAB fosmids: SAB5A16, SAB16A18, SAB16E6, SAB18J4 and SAB28M4, contained 31, 19, 31, 16 and 20 kbp of metagenomic material, respectively, whilst the LAB fosmids:, LAB4P4, LAB8M16, LAB9D24 and LAB9P23, contained of 28, 39, 33 and 18 kbp of metagenomic material, respectively.
The protein coding sequences in fosmids SAB5A16, SAB16A18, SAB16E6, SAB18J4, SAB28M4, LAB4P4, LAB8M16 and LAB9P23 were more closely related to Prevotella ruminicola 23 and Bacteroides species. Coding sequences in fosmid LAB9D24 were most closely related to Butyrivibrio fibrisolvens, Ruminococcus sp., Bacteroides sp. and Prevotella sp. (Supplementary material Table S1-S9) Fourteen putative genes showing similarity to known esterase/lipase genes were retrieved and were named lip1 to lip14, two patatin-like phospholipase genes were also found and named pl1 and p12 (Table 1). No lipase genes were retrieved from LAB8M16 or LAB9D24, either because of the incomplete assembly of the fosmid sequence due to low sequence coverage, or possibly because the blue hue observed between 20 and 24 h in the spirit blue agar plate assay was a false positive.
DNA Sequencing and Sequence Analysis
The fosmid sequences were determined using a high throughput pyrosequencing GS FLX (454 Life Sciences) at Aberystwyth University, UK. The purified lipase-positive fosmids were fragmented to 600-900 by fragments by nebulisation. The sheared fosmids were ligated to molecular barcodes (Multiplex Identifiers, MID, Roche Life Sciences) containing short oligonucleotide adaptors “A” and “B”. This was in order to specifically tag each sample in the sequencing run. The MID-adaptor ligated DNA libraries were mixed in an equimolar amount and clonally amplified by emulsion PCR using 13.6×106 Sepharose beads per emulsion reaction. Emulsions were then broken with isopropanol and emulsion PCR beads were enriched for template-positive beads and bead-attached DNAs were denatured with NaOH and sequencing primers were annealed. Approximately 790,000 beads with clonally amplified DNA were then deposited on two of four 70×75 mm regions of the PicoTiterPlate. The PicoTiterPlate device was then loaded onto the 454 instrument along with the sequencing reagents, and sequences were obtained according to the manufacturer's protocol. An SFF (Standard Flowgram Format) file was obtained for each sample, and nucleotide sequence data and phred-like quality scores were extracted. The reads from each of the pooled libraries were identified by their MID tags by the data analysis software gsAssembler v2.5.3 (Roche Life Sciences) after the sequencing run. The assembly was done using the default parameters on gsAssembler. BLASTN on NCBI was used to trim the vector sequence from the contigs. The GC content was calculated with BioEdit. The open reading frames (ORFs) were characterized using ORF Finder (available at [http://www.ncbi.nlm.nih.gov/gorf/gorf.html]), BLASTN (non-redundant nucleotide collection) and BLASTP (non-redundant protein sequences database) on NCBI. The following criteria were applied to define ORFs:
Only sequences preferentially non-overlapping and encoding peptides longer than 50 amino acids were retained,
Where putative ORFs were found in different reading frames, only those with known homologs were retained,
In the case of various putative overlapping ORFs in different reading frames with no known homologs, the ORFs with the longest sequence were selected.
The theoretical molecular mass and isoelectric point of the deduced amino acid sequences were calculated using the Compute pI/MW tool on the ExPASy proteomics server (available at http://expasy.org/tools/pi_tool.html). Signal sequences for peptide cleavage were analyzed using SignalP 4.0 (Petersen et al., 2011) using the Gram-negative model. Conserved domains in the amino acid sequences were analyzed with Conserved-Domain search on NCBI (Marchler-Bauer et al., 2011) and the Pfam database (version 25.0, available at http://pfam.sanger.ac.uk/). Clustering analysis was conducted by carrying out multiple sequence alignments using the ClustalW online tool (available at http://www.ebi.ac.uk/Tools/msa/clustalw2/) for the protein sequences. Closely related homologs were identified from the NCBI non-redundant database using BLASTP searches. Sequences with alignment >50% identities and e-value <1e-10 were considered. As the classification of lipolytic enzymes is based on the comparison of their protein sequences (Arpigny and Jaeger, 1999, Haussmann and Jaeger, 2010), the protein sequences from 50 members representing eight lipolytic families were retrieved from NCBI (Liu et al., 2009). A multiple sequence alignment file was constructed using ClustalW online tool on the Pfam conserved domains (α/β hydrolase fold or esterase/lipase domain). MEGA5 software (Tamura et al., 2011) was used to construct the tree using the neighbor-joining method by following Dayhoff PAM matrix model.
BLASTN analysis showed that the gene sequences of lip1, lip2, lip3, lip4, lip5, lip6, lip9 and lip10 had some similarity to the gene o23 coding for an ester hydrolase in an uncultured marine prokaryote (AJ811969) (74 to 80% sequence identity). Genes lip7, lip12 and lip14 were related (70-72% sequence identity) to an esterase gene isolated from an uncultured bacterium from a cow rumen metagenome, whilst lip8 and lip11 were related (75% sequence identity) to an esterase/lipase gene retrieved from a phagemid clone from a bovine rumen metagenomic library (
Phylogenetic placement of the predicted proteins suggest that lipases from all the main lipase families described by Arpigny and Jaeger (1999), shown in
Lip8 and lip11 clustered with genes from family IV as defined by Arpigny and Jaeger (1999). This was confirmed by multiple alignments with proteins from this family (
Alignments indicated that lip1, lip2, lip5, lip6, lip9, and lip10 might be more closely related to family VII. Multiple amino acid alignment (
Lip13 clustered with so-called GDSL enzymes from family II. The active site motif GDS(L) was found in the N-terminus of the protein sequence and elements of the five blocks of conserved amino-acids were present in its sequence (
The dendrogram (
Pl1 and pl2 clustered with enzymes from family VIII; however, multiple amino acid alignments did not show conserved motifs characteristic from this enzyme family.
Preparation of Anaerovibrio Lipolytica 5S Genomic DNA
Pure cultures of A. lipolytica strain 5S, as first isolated by Hobson and Mann (1961) at the Rowett Research Institute (Aberdeen, Scotland), came from the Rumen Microbiology group collection at IBERS. The genomic DNA was extracted using the BIO101 FastDNAC) Spin Kit for Soil (Qbiogene, Cambridge, UK) from approximately 2 mg of cryopreserved freeze-dried culture. The manufacturer's guidelines were followed, with the exception that the sample was processed for 3×30 s at speed 6.0 in the FastPrep instrument (QBiogene), with incubation for 30 s on ice between bead-beating.
De novo Genome Sequencing
The draft nucleotide sequence of the bacterium was established by a shotgun sequencing approach carried out on a Genome Sequencer FLX system (454 Life Sciences, Roche), following the supplier's protocol. Assembly of the reads was accomplished using gsAssembler v2.5.3 software (Roche, Life Sciences), using the default parameters.
454 pyrosequencing generated 340,862 high quality reads with an average length of 425.16 bp, representing 144,706,594 by total information. These data represented 36×coverage for an estimated bacterial genome size of 4 Mbp. The assembly of the uncompleted draft genome resulted in 285 contigs with 2,830,874 by total sequence information, comprising 247 large contigs (>500 bp) with a total size of 2,816,384 bases. The RAST annotation identified 2,673 coding sequences and the G+C content was 43.28%. Copies of the 5S and 23S rRNA genes (6 and 1 respectively) and 60 predicted tRNA genes were identified within the genome. There are 268 subsystems represented in the genome, however 63% of the predicted genes could not be assigned to a subsystem. Two genes annotated as “GDSL family lipolytic enzyme” and one gene annotated as “carboxylesterase” were named alipA, alipB and alipC respectively.
Annotation and Sequence Analysis
The contigs were submitted for genome annotation to the RAST server at http://rast.nmpdr.org (Aziz et al., 2008), tRNAscan-SE 1.23 (Lowe and Eddy, 1997) and RNAmmer 1.2 (Lagesen et al., 2007).
The predicted lipase genes and amino acid sequences were compared for similarity to known sequences using BLASTN and BLASTP search. Their signal sequences for peptide cleavage were predicted using SignalP 4.0 (Petersen et al., 2011). CD search (Marchler-Bauer et al., 2011), the Pfam database (version 25.0, available at http://pfam.sanger.ac.uk/) and ClustalW (Thompson et al., 1994) were used to search for conserved domains in the predicted amino acid sequences and to execute multiple alignments to find potential gene products relatedness to known families of lipolytic enzymes. The theoretical molecular mass and isoelectric point of the deduced lipolytic protein sequences were calculated using the Compute pI/Mw tool on the ExPASy proteomics server (available at http://expasy.org/tools/pi_tool.html, May 2011).
The genes length varied from 744 to 1,476 bp; and features of the encoded proteins are presented in Table 2. Tables 3 and 4 present the results of the BLASTN and BLASTP analysis of the identified putative lipase genes.
Gene alipA matched with a gene coding for a lipolytic protein from Selenomonas sputigena, with 63% identity, whereas no homologous sequences were found for genes alipB and alipC in the Genbank database. However, the proteins were found to match with proteins from various Veillonellaceae, and the best hits were with GDSL lipolytic proteins from Selenomonas species for the proteins alipA and alipB (56 and 41% identity respectively) and with a lipase/esterase from Mitsuokella multiacida for alipC (51% identity). AlipC also shared 42% amino acid identity (e-value 8e-50) with the lipase from rumen metagenome RlipE2 (Liu et al., 2009).
Anaerovibrio lipolytica 5S
sputigena ATCC 35185
Selenomonas sputigena
Selenomonas sp. oral
Mitsuokella multiacida
Expression and Purification of Recombinant Lipases
Primers for the amplification of the lipase genes were designed with FastPCR 6.1 (Kalendar et al., 2009), with and without the N-terminal signal sequence where one could be identified. The PCR reaction was set up in a total volume of 25 μl as follows: 2 μL of template (˜100 ng), 1 μl of forward and reverse primer (10 μM), 8.5 μl of molecular water and 12.5 μl of PCR mastermix (ImmoMix™, Bioline UK Ltd., London, UK). Initial activation of the Taq was performed for 10 min at 95° C., followed by 25 cycles as follows: 95° C. for 30 s, 50° C. for 30 s, 72° for 2 min, followed by a final extension at 72° C. for 8 min and holding of samples at 4° C. After PCR, the products were verified by electrophoresis on a 1% agarose gel using a 1 kb ladder. The band of interest was cut out with a sterile razor blade and the DNA eluted using the MinElute Gel Extraction kit (Qiagen, Crawley, UK).
Anerovibrio
lipolytic
F, forward primer; ssF, forward primer with predicted signal sequence removed; R, reverse primer
The expression of the lipolytic genes was then undertaken using the pTrcHis TOPO® TA Expression kit (Invitrogen, Carlsbad, Calif., USA) following the supplier's protocol. The PCR product was ligated to the pTrcHis TOPO vector and transformed into E. coli TOP10 cells. Twelve colonies for each transformation were picked for secondary screening and their insert was analyzed for size and orientation by tip-dip PCR using the gene specific forward primer and the vector specific pTrcHis reverse primer (5′-GATTTAATCTGTATCAGG-3′).
Proteins from the rumen metagenome were purified from 50 ml cultures in the presence of 50 μg/ml ampicillin on 2% inoculation from 2 ml of LB starter culture containing 50 μg/ml ampicillin (inoculated with a single colony) and grown overnight at 37° C. with shaking. The culture was induced with 1 mM IPTG after growth to mid-log growth phase and grown with shaking for a further 5 h at 37° C. The cells were then harvested by centrifugation at 3000×g, 10 min, 4° C., and the pellets stored at -80° C. before proceeding to protein purification. Purification of the proteins was carried out in native conditions using the ProBond™ Purification System (Invitrogen, Carlsbad, Calif., USA). Protein concentrations were estimated using the Bradford procedure (Bradford, 1976) employing BSA as the standard (Sigma, Dorset, UK).
Protein expression of A. Lipolytica lipases was accomplished by growing and inducing 50 ml of cells as follows: 2 ml of LB broth containing 50 μg/ml ampicillin were inoculated with a single colony and grown overnight at 37° C. with shaking. Subsequently, 50 ml of LB broth containing 50 μg·ml-1 ampicillin were inoculated with 1 ml of the overnight culture and grown until mid-log. The culture was then induced with IPTG to a final concentration of 1 mM and the culture grown at 37° C. with shaking at 100 rpm for 5 h. The cells were then harvested by centrifugation at 3000×g, 10 min, 4° C., and the pellets stored at -80° C. before proceeding to protein purification. Purification of the proteins was carried out in native conditions using the ProBond™ Purification System (Invitrogen, Carlsbad, Calif., USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the success of the purification.
Protein concentration was estimated using the Bradford procedure (Bradford, 1976) employing BSA as the standard (Sigma, Dorset, UK). The enzyme sample (5 μl) was mixed with 250 μl of Bradford reagent in a microplate, the plate was shaken for 30 s and incubated at room temperature for 20 min. The formation of the blue-coloured Coomassie-Blue G-250 complex was then monitored at 595 nm on a PowerWave XS microplate reader (BioTek Instruments Inc., Potton, UK).
Enzymatic Assays
Enzyme activity was quantified on a temperature-controlled Powerwave XS microplate reader (BioTek Instruments Inc., Patton, UK) based on the level of ρ-nitrophenol released following the hydrolysis of ρ-nitrophenyl ester substrates by the enzyme (Lee et al., 1993; Pinsirodom and Parkin, 2001). The production of ρ-nitrophenol was monitored in triplicates every minute for 10 min at 410 nm, and data were collected with the software Gen5 v1.10 (BioTek Instruments Inc., Potton, UK). Unless otherwise described, enzyme activity was measured by a standard assay at 39° C., with 1 mM ρ-nitrophenyl ester substrates in 50 mM morpholineethanesulfonic acid (MES, pH 6.5) containing 1% acetonitrile. The substrates used in standard conditions were ρ-nitrophenyl caprate (C10) for lip4 and lipl3ss, ρ-nitrophenyl caproate (C6) for lip6 and ρ-nitrophenyl caprylate (C8) for pl1 and pl2ss, alipA, alipBss and alipC. After pre-incubation for 3 min, the reaction was started by the addition of 2 μl of the eluted fraction of purified enzyme (˜0.4 mg/ml). Blank reactions were performed with every measurement to subtract appropriate values for nonenzymatic hydrolysis of the substrate. One unit of enzyme activity was defined as the amount of activity required to release 1 pmol of ρ-nitrophenol/min from ρ-nitrophenyl ester.
Substrate Specificity
The following ρ-nitrophenyl esters with different chain length were used at 1 mM final concentration for assaying substrate specificities: ρ-nitrophenyl butyrate (C4), ρ-nitrophenyl caproate (C6), ρ-nitrophenyl caprylate (C8), ρ-nitrophenyl caprate (C10), ρ-nitrophenyl laurate (C12), ρ-nitrophenyl myristate (C14), ρ-nitrophenyl palmitate (C16) and p-nitrophenyl stearate (C18). The ρ-nitrophenyl ester substrates with C4 to C10 acyl chains were dissolved in acetonitrile at a concentration of 100 mM. P-nitrophenyl ester substrates with C12 to C18 acyl chains were dissolved in a 1:4 mixture of acetonitrile and 2-propanol in order to solubilise the substrate, and reactions were performed with final concentrations of 1% acetonitrile and 4% 2-propanol.
To examine substrate specificity, activity was tested against various ρ-nitrophenyl esters with different acyl chain lengths. The results under standard assay conditions of pH 6.5 and 39° C. are presented in Table 7.
Lip4 and lipl3ss showed narrow chain length specificity, with the highest specific activity against ρ-nitrophenyl laurate (373.4 and 398.6 U/mg respectively) and a lower specific activity against ρ-nitrophenyl caprate (107.7 and 214.6 U/mg respectively); activity against other substrates was very low or not detected.
Protein lip6 exhibited the typical behavior for carboxylesterases, showing a preference for short acyl chains. The highest specific activity was observed with ρ-nitrophenyl butyrate (273.3 U/mg) and activity values decreased with the increase in the acyl chain length. No detectable activity was observed against ρ-nitrophenyl stearate.
Pl1 showed a broader range of activity with higher specific activities against short to medium acyl chain length: the activities were respectively 247.8 U/mg with ρ-nitrophenyl butyrate, 317.5 U/mg with ρ-nitrophenyl caprylate, 224.6 U/mg with ρ-nitrophenyl caprate and laurate.
Pl2ss showed no identifiable substrate preference. AlipA and alipBss showed a narrow chain length specificity, with the highest specific activity against ρ-nitrophenyl laurate (640 U·mg-1) and myristate (157 U·mg-1) respectively, and lower specific activity against ρ-nitrophenyl caprate (33 and 43 U·mg-1 respectively). AlipC showed a broader range of activity with higher specific activities against short to medium acyl chain length: the activities were 187 U·mg-1 against ρ-nitrophenyl butyrate, 270 U·mg-1 against ρ-nitrophenyl caprylate, 118 and 242 U·mg-1 against ρ-nitrophenyl laurate.
Effect of pH on Enzyme Activity
The effect of pH on the activity of the enzymes was examined across the pH range 3.5 to 10.0 using a wide-range pH buffer (Cai et al., 2011), containing 40 mM each of acetic acid, MES, N-(2-hydroxyethyl) piperazine-N′-ethanesulfonic acid (HEPES), N-[Tris(hydroxymethyl) methyl]-3-ami nopropanesulfonic sodium salt (TAPS) and N-cyclohexyl-3-aminopropane sulfonic acid (CAPS). The pH was adjusted by adding 1 M HCl or 1 M NaOH as appropriate at 39° C. The specific activity of the enzyme was determined spectrophotometrically at 348 nm as it is the pH-independent isobestic wavelength of ρ-nitrophenoxide and ρ-nitrophenol (Hotta et al., 2002).
The pH assays were carried out using ρ-nitrophenyl caprate (C10) as the substrate for lip4 and lipl3ss, ρ-nitrophenyl caproate (C6) for lip6 and ρ-nitrophenyl caprylate (C8) for pl1 and pl2ss, at a constant temperature of 39° C. in a wide-range pH buffer set at the indicated pH values.
Proteins Lip4, lip6, lipl3ss and pl1 had maximal activity at neutral or slightly alkaline pH (7 or 7.5). Lip6, lipl3ss and pl1 exhibited >50% activity in the pH range of 6.5-8.0, while lip4 showed activity over a broader pH range as it presented 53% of its maximum activity level at pH 10. Pl2ss had optimum pH at 8.5 respectively and presented activity >50% in alkaline pH range 8.5-10.0.
AlipA and alipC had maximal activity at pH 8.5 and 9.0 respectively, and presented >50% activity in alkaline pH ranges, respectively 7.5-9.5 and 9.0-10.0. AlipBss showed >50% of maximum activity in the pH range 6.0-8.0, with maximal activity at pH 7.5.
Effect of Temperature on Enzyme Stability and Thermostability
The effect of temperature on the activity of enzyme activity was examined across the range 25-70° C. under standard assay conditions. The pH of the MES buffer was adjusted to 6.5 at respective temperatures. The thermostability of the enzymes was analyzed by measuring the residual activity after incubating the enzyme (2 μl in 50 mM MES, pH 6.5) for 1 h at 50, 60 and 70° C.
The assays were carried out using ρ-nitrophenyl caprate (C10) as the substrate for lip4 and lipl3ss, ρ-nitrophenyl caproate (C6) for lip6 and ρ-nitrophenyl caprylate (C8) for pl1 and pl2ss, at a constant pH of 6.5 in 50 mM MES.
The optimum temperatures were determined as 40° C. (lip4, lip6, lipl3ss), 45° C. (pl1), and 30° C. (pl2ss). The temperature range where the enzyme retained more than 50% activity was narrow for lip4 (around 40° C.), pl1 (45-50° C.) and lipl3ss (around 40° C.), while it was broader for lip6 (40-50° C.) and pl2ss (25-40° C.). The temperature stability of the proteins was examined by measuring residual activity after incubating the purified enzymes for 1 h at 50, 60 or 70° C. as shown in Table 8. The proteins lip4, lipl3ss, and pl1 appeared to be temperature sensitive as less than 50% of activity was measured after 1 h incubation at 50° C. Activities ranged from 8 to 45% after incubating at 60 or 70° C. Lip6 appeared to have some thermostability: at 50° C., it had 57.9% activity but lost activity after incubation at 60 or 70° C. Only the protein pl2ss displayed some thermostability as it displayed nearly 90% of its activity after incubation at 50 and 60° C., yet lost 30% of its activity after incubation at 70° C.
The optimum temperatures were determined as 40° C. (alipA, alipC) and 55° C. (alipBss). The temperature range where the enzyme retained more than 50% activity was 40-50° C. for alipA, 35-55° C. for alipBss, and 35-50° C. for alipC. The temperature stability of the proteins was examined by measuring its residual activity after incubating the purified enzymes for 1 h at 50, 60 or 70° C. (Table 8). The proteins alipBss and alipC appeared to be temperature sensitive as less than 50% of activity was measured after 1 h incubation at 50° C. Activities ranged from 8 to 45% after incubating at 60 or 70° C. AlipA appeared to have some thermostability: it retained around 50% activity after incubation at 60 and 70° C.
Effect of Metal Ions on Enzyme Activity
The effect of metal ions on the activity of the enzymes were investigated by incubating the enzymes with various metal chloride salts (Na+, K+, NH4+, Mg2+, Ca2+, Mn2+, Zn2+, Co2+) at final concentrations of 5 mM in 50 mM MES (pH 6.5) for 30 min at room temperature. The remaining activity was then measured under standard assay conditions. The results are presented in Table 9.
Lip4 activity was strongly inhibited by NH4+, Mg2+, Ca2+, Zn2+ and Co2+ (residual activities <55%), moderately inhibited by Na+ and K+ (residual activities, 70% and 88% respectively), and no effect of Mn2+ was observed. Lip6 activity was totally inhibited by Mn2+ and Co2+ (residual activities, 8 and 0% respectively), and strong inhibition was also observed in the presence of K+, NH4+ and Mg2+ (residual activity <62%). Ca2+ and Zn2+ moderately inhibited lip6 activity (residual activities, 76 and 84% respectively) while Na+ slightly increased its activity (101%). Only Zn2+ had a strong inhibitory effect on pl1 activity (15% residual activity) while Na+, K+, NH4+, Mg2+ and Co2+ had moderate inhibitory effects (70% to 76% residual activities). Ca2+ and Mn2+ activated pl1 (residual activities, 163 and 124% respectively). Lip13ss activity was strongly inhibited in the presence of K+, Mg2+, Ca2+, Zn2+ and Co2+ (residual activities <50%) and more moderately by Na+, NH4+ and Mn2+ (residual activities from 61 to 90%). Pl2ss was totally inhibited in the presence of Ca2+ and strongly with Co2+ (57%), but its activity increased significantly with other ions (from 115% with Mn2+ to up to 368% in the presence of Zn2+).
Ca2+, Mn2+ and Co2+ inhibited alipA activity(<68% activity), K+ and NH4+ did not modify the activity (98-99% activity), while Na+, Mg2+ and Zn2+ activated alipA. AlipBss activity was strongly inhibited by Ca2+ and Mg2+ (residual activities, 33 and 56% respectively) and moderately inhibited by Na+, K+ and NH4+ (residual activities, 72, 67 and 69% respectively), whereas Mn2+, Zn2+ and Co2+ activated alipBss (residual activities, 130, 107, and 103% respectively). Zn2+ strongly inhibited alipC activity (17% residual activity); NH4+, Mg2+ and Co2+ had more moderate inhibitory effects (residual activities from 74 to 81%). K+ had no effect, while Na+, Ca2+ and Mn2+ activated alipC (residual activities, 119, 185, and 141% respectively).
Formation of Polylactic Acid Polymer (PLA)
Lactic acid polymerizing assays as described by Lassalle et al., (2008); Lassalle and Ferreira, (2008) were performed using equivalent concentrations of our rumen derived (discovered using functional metagenomics) lipases designated as Lipase B and Lipase F (Table 10; this table also provides information on our other rumen metagenomically derived lipases which have been biochemically characterised) and Novozym 435 as a positive control.
In order to isolate the newly formed polylactic acid, dichloromethane was added to the solutions, followed by dH2O. At the interface of toluene and water, the synthesized PLA could be observed. The solutions were then frozen at -20° C. and freeze dried for 2-3 days, in order to isolate the polymer. FTIR was conducted in order to check PLA formation in comparison to Novozym 435. Freeze-dried weight was also recorded in order to assess comparative yield. Our data show that both of our lipases produced approximately 10% more PLA than novozym 435 under our test conditions and FTIR confirmed this data whilst illustrating that we had indeed made PLA. The chemical reaction in the formation of PLA is as follow:
Thus the presence of C—O—C linkages increase as polymerization proceeds. Indeed in our FT-IR traces of isolated putative PLA we see characteristic ester absorption peaks at approx. 1700, 1670 for the stretching vibration of the —COO— and at 1100 and 1200 for the stretching vibration of the C—O—C, proving the production of PLA (
Nucleotide Sequence Accession Numbers
The nucleotide sequences of the genes reported here are available in the Gen Bank database under accession numbers: alipA, KC579357; alipB, KC579358; alipC, KC579359; lip1, JX469447; lip2, JX469448; lip3, JX469449; lip4, JX469450; lip5, JX469451; lip6, JX469452; lip7, JX469453; lip8, JX469454; lip9, JX469455; lip10, JX469456; lip11, JX469457; lip12, JX469458; lip13, JX469459; lip14, JX469460; pl1, JX469461; and pl2, JX469462.
It will be appreciated by those skilled in the art that the foregoing description is exemplary and explanatory in nature, and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, an artisan will recognise apparent modifications and variations that may be made without departing from the spirit as scope of the invention as defined in the appended claims.
Mølgaard, A., Kauppinen, S., Larsen, S. (2000) Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Structure 8(4):373-383.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1216482.8 | Sep 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2013/052414 | 9/16/2013 | WO | 00 |
