WO2014074352A1 - Dieselzymes and methanol tolerant lipases - Google Patents

Abstract

This disclosure relates to modified lipases. The disclosure further relates to cells expressing such modified lipases and methods of producing biodiesel products by contacting a suitable substrate with such cells.

Description

DIESELZYMES AND METHANOL TOLERANT LIPASES

CROSS REFERENCE TO RELATED APPLICATIONS

[ 0001 ] The application claims priority to U.S. Provisional Patent

Application No. 61/722,786, filed November 6, 2012, the disclosure of which is incorporated herein by reference.

STATEMENTS REGARDING FEDERALLY SPONSORED RESEARCH

[ 0002 ] This invention was made with Government support of Grant

No. DE-FC02-02ER64321, awarded by the United States Department of Energy. The Government has certain rights in this invention.

TECHNICAL FIELD

[ 0003 ] This invention relates to modified lipases. The invention further relates to cells expressing such modified lipases and methods of producing biofuels products by contacting a suitable substrate with such modified lipases and/or such cells.

BACKGROUND

[ 0004 ] Lipases (triacylglycerol acylhydrolases , E.C. 3.1.1.3) consist of a genetically diverse and distinctive grouping of water- soluble hydrolytic enzymes that typically act on the ester bonds of lipid substrates. Lipids can include fats, waxes, sterols, fat- soluble vitamins, monoglycerides , diglycerides , fatty acyls, polyketides, and fatty acids, and exist as a number of variations containing different additional chemical structures such as

phospholipids, glycerolipids , glycerophospholipids , sphingolipids , sterol lipids, prenol lipids, saccharolipids , etc. Lipases have been used in ester and amide synthesis, kinetic resolutions or asymmetric synthesis to obtain optically pure compounds, and lipid modifications

(Bornscheuer and Kazlauskas, 2005) . Lipases play an essential role in: (1) the metabolism of dietary lipids, (2) injury and

inflammation, (3) cell biology, (4) fermentation, (5) biocatalysis ,

(6) vitamin absorption, (7) laundering, (8) synthesis of

pharmaceuticals and many other biological and chemical processes. Such wide and varying roles have been attributed to lipase stability in organic solvents, high specificity, high enantio-selectivity and regio-selectivity, and a general lack of a need of cofactors for their action. Genes encoding lipases have been found in most, if not all, types of organisms. SUMMARY

[ 0005 ] The disclosure provides an isolated or recombinant lipase polypeptide comprising a sequence that is at least 80% identical to SEQ ID NO : 2 and having at least one mutation at a residue selected from the group consisting of R33, L64, A70, G181, G202, K208, S238, G266 and any combination thereof and wherein the polypeptide has lipase activity and improved methanol tolerance and/or

thermostability compared to a wild-type lipase of SEQ ID NO: 2. In one embodiment, the polypeptide has a mutation at G181 and S238 and has improved thermostability compared to a polypeptide comprising SEQ ID NO : 2. In a further embodiment, the mutation at G181 is G181C. In yet a further embodiment, the mutation at S238 is S238C. In yet another embodiment of any of the foregoing the polypeptide is at least 80% identical to SEQ ID NO: 15 and having C181 and C238 mutation. In yet a further embodiment, the polypeptide is at least 85%, 95% or 100% identical to SEQ ID NO: 15 and having C181 and C238. In yet another embodiment, the the polypeptide further comprises a mutation at a residue selected from the group consisting of R33, L64, A70, G202, K208, G266 or any combination thereof and wherein the lipase has improved methanol tolerance compared to a polypeptide of SEQ ID NO : 2. In a further embodiment, the polypeptide further comprises a mutation selected from the group consisting of R33T, L64I, A70T, G202E, K208N, G266S and any combination thereof. In still a further embodiment, the polypeptide comprises the mutations R33T, L64I, A70T, G202E or G202D, K208N, and G266S. In yet another embodiment, the polypeptide further comprises a mutation at a residue selected from the group consisting of L13, N17, F40, V61, E67, A70, S95, R117, M119, T138, S141, S144, G145, D149, A153, G206, E207, N223, F225, G243, L245, 1255, Y267, D270, V272, Q277 and any combination thereof. In yet another embodiment, the polypeptide has mutations selected from the group consisting of: (a) F225L and Q277L;

(b) N17S, M119I, F225L, D270N and Q277L; and (c) N17S, E67A, M119I, A153V, F225L, I255F, D270N and Q277L. In another embodiment, the polypeptide is at least 80% identical to SEQ ID NO:2 and has mutations selected from the group consisting of: (a) L64I, A70T, G181C, K208N, F225L, S238C and Q277L; (b) N17S, L64I, A70T, M119I, G181C, G202E, K208N, F225L, S238C, G266S, D270N and Q277L; and (c) N17S, R33T, L64I, E67A, A70T, M119I, A153V, G181C, G202E, K208N, F225L, S238C, I255F, G266S, D270N and Q277L. In yet a further embodiment, the polypeptide comprises a sequence as set forth in SEQ ID NO: 47. In yet a another embodiment, the polypeptide comprises a sequence as set forth in SEQ ID NO: 65. In yet another embodiment, the polypeptide comprises a sequence as set forth in SEQ ID NO: 85. In yet another embodiment, the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, and 85. In yet another embodiment of any of the foregoing, the polypeptide further comprising from 1-20 conservative amino acid substitutions.

[ 0006 ] The disclosure also provides an isolated or recombinant polynucleotide encoding any of the foregoing polypeptides. In one embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, and 84. The disclosure also provides a vector comprising the polynucleotide of any of the foregoing. In one embodiment, the vector is an expression vector.

[ 0007 ] The disclosure also provides a host cell transfected or transformed with a vector or polynucleotide of the disclosure as described above. The host cell can be prokaryotic or eukaryotic.

[ 0008 ] The disclosure also provides a method of producing a fatty acid from a tri-, di- or mono-glyceride comprising contacting the tri-, di- or mono-glyceride with a polypeptide as described above under conditions wherein a fatty acid methyl ester is produced.

[ 0009 ] The disclosure also provides a method of producing a fatty acid from a tri-, di- or mono-glyceride comprising contacting the tri-, di- or mono-glyceride with a host cell that produces a

polypeptide of the disclosure under conditions wherein a fatty acid methyl ester is produced.

[ 0010 ] The disclosure provide polypeptide produced from directed evolution that have improved properties for biodiesel production. One evolved enzyme, Dieselzyme 4, has better methanol tolerance and heat stability than the top industrially used lipase from B. cepacia. Additionally, Dieselzyme 4 catalyzes efficient synthesis of biodiesel even in the presence of a high concentration of water. Unlike the B. cepacia lipase Dieselzyme 4 is highly expressed in an active form in E. coli, so it remains a viable platform for further engineering efforts to develop better catalysts for biodiesel production in the future .

[ 0011 ] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

[ 0012 ] Figure 1 shows a general transesterification reaction catalyzed by lipases.

[ 0013 ] Figure 2A-D shows: (A) a ribbon structure of P. mirabilis lipase (PML) . Lid helices, bound Ca²⁺, and catalytic triad as a ball and stick are depicted. The location of the equivalent disulfide bond found in the B. cepacia lipase, but absent in PML, is labeled and shown as sticks. (B) Thermal inactivation as a function of

temperature. Residual activity was measured after incubation for 1 hour. (C) Inactivation as a function of methanol concentration.

Residual activity was measured after incubation for 2 hours after dilution to 10% methanol. (D) Inactivation by 50 % methanol over time. Samples were diluted to 10% methanol prior to assay for residual activity as in (C) . Results are the average of three independent experiments. WT PML (black circles), BCL (open circles), and Dieselzyme 1 (squares) .

[ 0014 ] Figure 3A-C shows a schematic of the screening protocol for improved methanol tolerance by directed evolution of PML. (A) Steps for colony lift assay and staining protocol. (B) Hydrolysis of 1-napthyl palmitate and detection of 1-naphthol by fast blue to assay colonies for residual activity after exposure to methanol and heat. (C) Representative filter following step 9 in (A) . Black arrows point to positives identified on the filter for futher analysis.

[ 0015 ] Figure 4A-B shows (A) thermal inactivation as a function of temperature. Residual activity was measured after incubation for 1 hour. (B) Thermal inactivation by incubation at 50 °C over time.

Results are the average of 3 independent experiments. WT PML (black circles) , BCL (open circles) , and Dieselzyme 1 (squares) from Figure 2 with mutants Dieselzyme 2 (diamond) , 3 (triangle) , and 4 (inverted triangle) .

[0016] Figure 5A-C shows (A) methanol induced inactivation as a function of methanol concentration. Samples were incubated for 2 hours and assayed after dilution to 10% methanol. Results are the average of 3 independent experiments. (B) Methanol induced

inactivation as a function of time. Samples were incubated in 50% methanol for 24 hours. At indicated time points, samples were diluted to 10% methanol and assayed for residual activity. Results are the average of 3 independent experiments. WT PML (black circles), BCL

(open circles) , and Dieselzyme 1 (squares) from Figure 2 with mutants Dieselzyme 2 (diamond) , 3 (triangle) , and 4 (inverted triangle) . (C) Inactivation profile of Wild-Type PML (black bar) , Dieselzyme 4

(bar), and BCL (white bar) in response to various solvents. Enzymes were incubated for 24 hours in 70% solvent and diluted to 10% prior to assaying for residual activity. Results are the average of three independent experiments.

[0017] Figure 6 shows operational stability of covalently immobilized Wild-Type PML, Dieselzyme 4, and BCL using a single addition of methanol. For each, the same amount of initial activity was added such that >40% conversion was seen over the first four hours: 0.25 g immobilized WT PML; 0.25 g immobilized Dieselzyme 4, or 0.1 g BCL. Reaction conditions: 1.5 mL canola oil, 312.5 L methanol

(5:1 MeOH:oil), 312.5 L 0.1 M sodium phosphate pH 7.0 (20% v/v with oil); temperature 25 °C; 180 rev/min. During cycles 1, 4, and 7, 100 L samples were taken at 0, 2, and 4 hours to monitor rate of reaction. After each 20 hour cycle, beads were washed with buffer followed by hexanes prior to addition of substrates and buffer. Data is the average of two experiments.

[ 0018 ] Figure 7A-E shows structure of PML mutant Dieselzyme 4.

(A) Cartoon representation of Dieselzyme 4. The locations of clustered mutations described in the text, designated Region 1 and Region 2, are highlighted with a boxs respectively. Mutations in Dieselzyme 4 are labeled and shown as sticks. Lid helices a5 and a8 are shown for clarity. The catalytic triad is shown as orange ball and stick. (B) 90° rotation of (A) . (C) Zoom in of Region 1 as seen in the wild-type PML structure. (D) Zoom in of Region 1 as seen in the Dieselzyme 4 structure. Mutations are highlighted as sticks. New H-bonds present in Dieselzyme 4 are shown as dashed black lines. (E) Zoom in of Region 2 as seen in the wild-type PML structure. The "helical loop" between residues 202 and 207 is highlighted orange. Potential H-bonds between residues R220 and H216 from helix a8, the loop region, and crystallographic waters are shown as dashes. The bound calcium is shown as a sphere. (F) Zoom in of Region 2 as seen in the Dieselzyme 4 structure. The remodeled "helical loop" between residues 202 and 207 is depicted. Residues G202E and E207 that form potential new salt-bridges with R220 and/or H216 from helix a8 are highlighted with a star. Other H-bonds are shown as dashes. The bound calcium is shown as a sphere.

[ 0019 ] Figure 8 shows an alignment of polypeptide sequences of the disclosure (SEQ ID NOs : 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, and 85) .

DETAILED DESCRIPTION

[ 0020 ] Before describing the invention in detail, it is to be understood that this invention is not limited to particular

compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a, " "an, " and "the" include plural referents unless the content clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the invention (s) , specific examples of appropriate materials and methods are described herein .

[ 0021 ] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are now described .

[ 0022 ] Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising"

"include," "includes," and "including" are interchangeable and not intended to be limiting.

[ 0023] It is to be further understood that where descriptions of various embodiments use the term "comprising, " those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of."

[ 0024 ] All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which are described in the publications, which might be used in connection with the description herein. However, with respect to any similar or identical terms found in both the

incorporated publications or references and those expressly put forth or defined in this application, then those terms definitions or meanings expressly put forth in this application shall control in all respects. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[ 0025] Biodiesel is a well validated transportation fuel that is an attractive alternative to petrodiesel. Biodiesel burns cleaner, releases less CO2 into the atmosphere, is biodegradable and can be obtained from renewable sources. Moreover, biodiesel can be used in existing diesel engines and is compatible with current

infrastructure. Finding new sources of biodiesel and improved production methods is therefore an important goal.

[ 0026] Biodiesel is typically composed of a mixture of fatty acid methyl esters (FAME), although other esters can be used (see, e.g, . Figure 1) . Biodiesel is usually synthesized via the base catalyzed transesterification of triacylglycerol (TAG) oils with methanol. In the base catalyzed reaction, the presence of water leads to the production of free fatty acids (FFAs) , which are a dead-end product. Moreover, any FFAs present in the oil cannot be converted to methyl esters. FFA contamination requires additional purification and also leads to emulsions that complicate reaction clean up. In addition, glycerol, the other value-added reaction product, becomes

contaminated with salt, lowering its value.

[ 0027 ] Lipases are a family of enzymes that are interesting as an alternative catalyst for biodiesel production. Like chemical catalysts, lipases can catalyze the transesterification of TAG with short chain alcohols at significant rates. However, unlike the base- catalyzed reaction, some lipases can also convert FFAs to FAMEs even in the presence of high levels of water. Moreover, because lipase catalysts can be easily separated, the glycerol product is not contaminated, increasing its potential as a value added product and lowering the net cost of biodiesel production.

[ 0028 ] An obvious drawback to the use of lipases is the high cost of the catalyst relative to a simple base such as NaOH. The initial investment cost of a lipase catalyst could be greatly mitigated, however, if it could be used for long periods of time and generate large quantities of biodiesel. Unfortunately, the presence of high concentrations of methanol used for biodiesel synthesis can cause significant loss of lipase activity due to alcohol induced

inactivation .

[ 0029 ] Various approaches have been employed to increase lipase lifetimes for biodiesel synthesis. Process engineering approaches have included enzyme immobilization, the use of co-solvents, or the step-wise feeding of alcohol so the concentration always remains low. However, the use of co-solvents further increase costs while stepwise feeding of methanol is difficult on an industrial scale. In addition, there have been efforts to identify natural lipases that are inherently more tolerant of the harsh conditions employed for biodiesel production.

[ 0030 ] Directed evolution and rational design of existing lipases has the potential to produce long lasting re-engineered enzymes with specific properties. For example, previous reports have shown that many α/β hydrolases, including lipases, could be successfully reengineered by directed evolution for improved thermostability as well as tolerance to a variety of polar solvents such as DMSO, DMF, and acetonitrile . To date, there are no reports of the reengineering of a lipase specifically for improved tolerance to short-chain alcohols such as methanol and ethanol that are used in biodiesel synthesis. The development of a lipase highly resistant to methanol inactivation would be especially useful as a catalyst for the economical synthesis of biodiesel.

[ 0031 ] Many microbial lipases have been identified as potential enzyme catalysts for biodiesel synthesis with a wide range of stabilities and catalytic efficiency. In spite of the large number of identified microbial lipases, most are poor targets for directed evolution methods because they require chaperones and/or post- translational modifications specific to the host organism. As a result most microbial lipases can only be produced in their native host, hampering engineering efforts. Indeed, one of the most widely used industrial lipases, LipA from Burkholderia cepacia, while highly active and tolerant to short-chain alcohols, requires a chaperone for proper folding and does not express solubly in E. coli.

[ 0032 ] Recently, an effective biodiesel-producing lipase from

Proteus sp . K107 was identified that can be expressed at high levels as a soluble protein in E. coli. The Proteus sp. K107 lipase is 100% identical to a lipase from Proteus mirabilis (referred to as Proteus mirabilis lipase (PML) ) . PML belongs to the Proteus/psychrophilic subfamily of 1.1 lipases which lack a leader sequence and disulfide bond present in other family 1.1 and 1.2 lipases. PML was shown to be tolerant to short-chain alcohols such as methanol and ethanol and could synthesize fatty acid methyl esters (biodiesel) in the presence of high concentration of water. A recombinant E. coli strain overexpressing PML could also be used as a whole cell biocatalyst for biodiesel production. PML, however, is irreversibly inactivated by incubation with more than 50% MeOH, a common weakness of lipases. The disclosure provides methods and development of a PML variant with significantly increased thermostability and tolerance to methanol that retains nearly wild-type activity at ambient temperature. A high-resolution crystal structure of the optimized lipase provides insight into adaptations in PML leading to methanol and heat tolerance . [ 0033] The "activity" of an enzyme is a measure of its ability to catalyze a reaction, i.e., to "function", and may be expressed as the rate at which the product of the reaction is produced. For example, enzyme activity can be represented as the amount of product produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants.

[ 0034 ] The term "coupling" means the ratio, in percent, of product formed to cofactor (NAD(P)H) consumed during the enzyme- catalyzed reaction. If 2 moles of cofactor are consumed for every mole of product made, then the coupling for the reaction is 50%.

Coupling figures provide the ability to assess how much cofactor is required to produce a given amount of product.

[ 0035] A polynucleotide, polypeptide, or other component is

"isolated" when it is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, synthetic reagents, etc.). A nucleic acid or polypeptide is "recombinant" when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid. For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example a non-naturally occurred variant of a naturally occurring gene, is recombinant. For example, an "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.

Typically, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[ 0036 ] The term "lipase activity" is defined herein as a triacylglycerol acylhydrolase activity (E.C. 3.1.1.3) which catalyzes the hydrolysis of a triacylglycerol to fatty acid(s). The substrate spectrum of lipases includes triglycerides, diglycerides , and monoglycerides , but for the purpose of the disclosure, lipase activity is determined using p-nitrophenyl butyrate as substrate. One unit of lipase activity equals the amount of enzyme capable of releasing 1 μΜ of butyric acid per minute at pH 7.5, 25 °C.

Encompassed within the term "lipase activity" are polypeptides that also have phospholipase activity, sterol ester esterase activity, and/or galactolipase activity, as defined herein.

[ 0037 ] A "mutant", "variant" or "modified" protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme,

polynucleotide, gene, or cell, that has been altered or derived, or is in some way different or changed, from a parent protein, enzyme, polynucleotide, gene, or cell. A mutant or modified protein or enzyme is usually, although not necessarily, expressed from a mutant polynucleotide or gene .

[ 0038 ] A "parent" protein, enzyme, polynucleotide, gene, or cell, is any protein, enzyme, polynucleotide, gene, or cell, from which any other protein, enzyme, polynucleotide, gene, or cell, is derived or made, using any methods, tools or techniques, and whether or not the parent is itself native or mutant. A parent polynucleotide or gene encodes for a parent protein or enzyme.

[ 0039 ] A "protein" or "polypeptide", which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. An "enzyme" means any

substance, composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions. A "native" or "wild-type" protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme,

polynucleotide, gene, or cell that occurs in nature.

[ 0040 ] The term "substrate" or "suitable substrate" means any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme catalyst. The term includes triacylglycerides , and includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate. Substrates for transesterification using the lipase enzymes of the disclosure include triacylglycerols and derivatives. The term "derivative" refers to the addition of one or more functional groups to a substrate .

[ 0041 ] As used herein the term "total turnover number" (TTN) is the total number of substrate molecules converted to product (or turned over) by a single enzyme over its lifetime or during a specified time period.

[ 0042 ] As will be described in more detail below, the disclosure is based, at least in part, on the generation and expression of enzymes that catalyze the transesterification of triacylglycerol to form fatty acid methyl esters. In one embodiment, polypeptides that have been engineered to perform this reaction are methanol resistant or have the ability to catalyze the reaction at higher concentrations of methanol compared to wild-type enzymes (e.g., parental

polypeptides from which the modified lipases are derived) . The ability of the polypeptides of the disclosure to have improved chemical and/or thermostability is based upon the mutation of amino acids in a parental polypeptide that modify the activity of the parental polypeptide .

[ 0043] A "mutation" means any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, gene, or cell. This includes any mutation in which a protein, enzyme, polynucleotide, or gene sequence is altered, and any detectable change in a cell or enzyme arising from such a mutation. Typically, a mutation occurs in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues. A mutation includes polynucleotide alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A mutation in a gene can be "silent", i.e., not reflected in an amino acid alteration upon expression, leading to a "sequence-conservative" variant of the gene. This generally arises when one amino acid corresponds to more than one codon.

[ 0044 ] While variants will be described in more detail below, it is understood that polypeptides of the disclosure may contain one or more modified or substituted amino acids. The presence of modified amino acids may be advantageous in, for example, (a) increasing polypeptide in vivo half-life, (b) reducing or increasing polypeptide antigenicity, and (c) increasing polypeptide storage stability. Amino acid(s) are modified, for example, co-translationally or post- translationally during recombinant production (e.g., N-linked glycosylation at N--X--S/T motifs during expression in mammalian cells) or modified by synthetic means.

[ 0045 ] Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a pegylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD- ROM (Humana Press, Towata, N.J.).

[ 0046 ] Recombinant methods for producing and isolating modified polypeptides of the disclosure are described herein. In addition to recombinant production, the polypeptides may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al .

(1969) Solid-Phase Peptide Synthesis (WH Freeman Co, San Francisco); and Merrifield (1963) J. Am. Chem. Soc. 85: 2149-2154; each of which is incorporated by reference) . Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide

Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer. [ 0047 ] The disclosure provides isolated or recombinant

polypeptides comprising residues 1 to about 861 of the amino acid sequence set forth in SEQ ID NO:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, or 85 as well as variants thereof. Such variants includes polypeptides having up to 50, 25, 10, or 5 conservative amino acid substitutions excluding residues identified in each of the recombinant polypeptides set forth in the following table (for example, with respect to PMR1.1A, 50, 25, 10, or 5 conservative amino acid substitutions can be present excluding residues 64 and 277) :

[ 0048 ] Table I: specific amino acid substitutions

Recombinant Mutant Residues

Polypeptide

PMR1 1A L64I/Q277L

PMR1 IB L64I/K208N/Q277L

PMR1 1C L64I/E207V/Q277L

PMR1 ID L64I/A70T/F225L/I255F/Q277L

DIESELZYME1 G181C/S238C

= PMR1. IE

PMR2 1A L64I/V64I/Q277L

PMR2 IB L64I/A70E/Y267C/Q277L

PMR2 1C L64I/S144T/Q277L

PMR2 IE L64I/N223S/Q277L

PMR2 IF L64I/Y267H/Q277L

PMR2 1G L64I/R117H/K208N/Q277L

PMR2 1H L64I/D149V/K208N/Q277L

PMR2 2A L64I/K208N/N223S/Q277L

PMR2 2B L64I/G181C/K208N/S238C

PMR2 2C L64I/G181C/K208N/N223S/S238C

PMR2 3A L61I/G181C/K208N/N223S/S238C/Q277L

PMR2 3B V61I/L64I/F143L/N223S/G243D/Q277L

PMR2 3C V61I/L64I/K208N/N223S/Q277L

PMR2 3D V61I/L64I/K208N/Q277L

PMR2 3E L64I/A70T/G181C/F225L/S238C/Q277L

DIESELZYME2 L64I/A70T/G181C/K208N/F225L/S238C/Q277L

= PMR2.3F

PMR3 1A N17D/L64I/A70T/G181C/F225L/S238C/Q277L

PMR3 IB L13F/L64I/A70T/G181C/F225L/S238C/Q277L

PMR3 1C L64I/A70T/G181C/F225L/S238C/D270N/Q277L

PMR3 2A L64I/A70T/G181C/F225L/S238C/G266S/Q277L

PMR3 2B L64I/A70T/M119I/G181C/G202E/F225L/S238C/G266S/Q277L

PMR3 2C L64I/A70T/ G181C/G202E/F225L/S238C/L245V/Q277L

PMR3 3A L64I/A70T/M119I/G181C/G202E/K208N/F225L/S238C/G266S/Q277L

PMR3 3B L64I/A70T/M119I/G181C/G202E/K208N/F225L/S238C/G266S/D270N/Q277L

DIESELZYME3 N17S/L64I/A70T/M119I/G181C/G202E/K208N/F225L/S238C/G266S/

D270N/Q277L

PMR4 1A N17S/L64I/E67A/A70T/M119I/A153V/G181C/G202E/K208N/F225L/S238C/

G266S/D270N/Q277L

PMR4 IB N17S/L64I/A70T/M119I/T138I/A153V/G145A/G181C/G202E/K208N/F225L

/S238C/I255F/G266S/D270N/Q277L

PMR4 1C N17S/L64I/A70T/S95N/M119I/G181C/G202E/K208N/F225L/S238C/G266S /D270N/V272I/Q277L

PMR4.ID N17S/R33T/L64I/A70T/M119I/G181C/G202E/K208N/F225L/S238C/G266S/

D270N/Q277L

PMR4. IE N17S/F40I/L64I/A70T/M119I/G181C/G202D/K208N/F225L/S238C/G266S/

D270N/Q277L

PMR4.IF N17S/L64I/A70T/M119I/S141A/G181C/G202E/K208N/F225L/S238C/G266S/

D270N/Q277L

PMR4.1G N17S/L64I/A70T/M119I/G181C/G202E/G206R/K208N/F225L/S238C/G266S/

D270N/Q277L

PMR4.2A L64I/E67A/A70T/M119I/A153V/G181C/G202E/K208N/F225L/S238C/I255F/

G266S/Q277L

PMR4.2B N17S/L64I/E67A/A70T/M119I/A153V/G181C/G202E/K208N/F225L/S238C/

I255F/G266S/D270N/Q277L

DIESELZYME4 N17S/R33T/L64I/E67A/A70T/M119I/A153V/G181C/G202E/K208N/F225L/

S238C/I255F/G266S/D270N/Q277L

[ 0049] "Conservative amino acid substitution" or, simply,

"conservative variations" of a particular sequence refers to the replacement of one amino acid, or series of amino acids, with a similar amino acid or amino acid sequences. One of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a percentage of amino acids in an encoded sequence result in "conservative variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.

[ 0050 ] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one conservative substitution group includes Alanine (A) , Serine (S) , and Threonine (T) . Another conservative substitution group includes Aspartic acid (D) and Glutamic acid (E) . Another conservative substitution group includes Asparagine (N) and Glutamine (Q) . Yet another conservative substitution group includes Arginine (R) and Lysine (K) . Another conservative substitution group includes

Isoleucine, (I) Leucine (L) , Methionine (M) , and Valine (V). Another conservative substitution group includes Phenylalanine (F) , Tyrosine

(Y) , and Tryptophan (W) .

[ 0051 ] Thus, "conservative amino acid substitutions" of a listed polypeptide sequence include substitutions of a percentage, typically less than 10%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative

substitution group. Accordingly, a conservatively substituted variation of a polypeptide of the disclosure can contain 100, 75, 50, 25, 10, 5, 3 or fewer substitutions with a conservatively substituted variation of the same conservative substitution group. Thus,

"conservative amino acid substitutions, " in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the polyeptides provided herein .

[ 0052 ] "Conservative variants" are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 30%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% similar, as determined according to an alignment scheme. As referred to herein, "sequence similarity" means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation. "Sequence identity" refers to the extent to which two nucleotide or amino acid sequences are invariant. Two polypeptides or polynucleotides of the disclosure can have at least 30%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. "Sequence alignment" means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity/identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN

algorithm, as well as BLASTN, BLASTP, and FASTA (Lipman and Pearson, 1985; Pearson and Lipman, 1988) . When using all of these programs, the preferred settings are those that results in the highest sequence similarity. For example, the "identity" or "percent identity" with respect to a particular pair of aligned amino acid sequences can refer to the percent amino acid sequence identity that is obtained by ClustalW analysis (version W 1.8 available from European

Bioinformatics Institute, Cambridge, UK) , counting the number of identical matches in the alignment and dividing such number of identical matches by the greater of (i) the length of the aligned sequences, and (ii) 96, and using the following default ClustalW parameters to achieve slow/accurate pairwise alignments--Gap Open Penalty: 10; Gap Extension Penalty: 0.10; Protein weight matrix:

Gonnet series; DNA weight matrix: IUB; Toggle Slow/Fast pairwise alignments=SLOW or FULL Alignment.

[ 0053 ] Two sequences are "optimally aligned" when they are aligned for similarity scoring using a defined amino acid

substitution matrix (e.g., BLOSUM62) , gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well- known in the art and described, e.g., in Dayhoff et al . (1978) "A model of evolutionary change in proteins" in "Atlas of Protein

Sequence and Structure," Vol. 5, Suppl . 3 (ed. M. 0. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. and Henikoffet al. (1992) Proc. Nat Ί . Acad. Sci . USA 89: 10915-10919 (each of which is incorporated by reference) . The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al . (1997) Nucl . Acids Res. 25: 3389-3402 (incorporated by reference herein), and made available to the public at the National Center for Biotechnology Information (NCBI) Website (www.ncbi.nlm.nih.gov). Optimal

alignments, including multiple alignments, can be prepared using, e.g., PSI-BLAST, available through the NCBI website and described by Altschul et al. (1997) Nucl. Acids Res. 25:3389-3402 (incorporated by reference herein) .

[ 0054 ] With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds to" the position in the reference sequence with which the residue is paired in the alignment. The "position" is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. For example, in SEQ ID NO:2, position 1 is M, position 2 is S, position 3 is T, etc. When a test sequence is optimally aligned with SEQ ID NO: 2, a residue in the test sequence that aligns with the T at position 3 is said to "correspond to position 3" of SEQ ID NO : 2. Owing to deletions, insertion, truncations, fusions, etc., that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence as determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[ 0055 ] Non-conservative modifications of a particular polypeptide are those which substitute any amino acid not characterized as a conservative substitution. For example, any substitution which crosses the bounds of the six groups set forth above. These include substitutions of basic or acidic amino acids for neutral amino acids, (e.g., Asp, Glu, Asn, or Gin for Val, lie, Leu or Met), aromatic amino acid for basic or acidic amino acids (e.g., Phe, Tyr or Trp for Asp, Asn, Glu or Gin) or any other substitution not replacing an amino acid with a like amino acid. Basic side chains include lysine (K) , arginine (R) , histidine (H) ; acidic side chains include aspartic acid (D) , glutamic acid (E) ; uncharged polar side chains include glycine (G) , asparagine (N) , glutamine (Q) , serine (S) , threonine (T) , tyrosine (Y) , cysteine (C) ; nonpolar side chains include alanine (A) , valine (V), leucine (L) , isoleucine (I), proline (P) , phenylalanine

(F) , methionine (M) , tryptophan (W) ; beta-branched side chains include threonine (T) , valine (V), isoleucine (I); aromatic side chains include tyrosine (Y) , phenylalanine (F) , tryptophan (W) , histidine (H) .

[ 0056 ] Accordingly, some amino acid residues at specific positions in a polypeptide are "excluded" from conservative amino acid substitutions. Instead, these restricted amino acids are set forth in Table 1, above, for a specific recombinant polypeptide. The "excluded" polypeptides for a particular recombinant polypeptide are identified as mutant residues. Accordingly, a polypeptide provided herein can include amino acids that are "restricted" to particular amino acid substitutions. It is understood that not all of the identified restricted residues need be altered in the same

polypeptide. In some embodiments, the disclosure encompasses polypeptides where only about 80%, 85%, 90% or 95% of the restricted amino acid residues are altered in a given polyeptide .

[ 0057 ] Accordingly, in some embodiments, a polypeptide provided herein includes amino acid residue substitutions that correspond to positions in a particular sequence at least 80%, 85%, 90%, or 95% of the time. In other words, the disclosure encompasses polypeptides that contain the recited amino acid substitutions at 80%, 85%, 90%, or 95% of the recited positions in a given sequence. The skilled artisan will recognize that not every substitution from a group of substitutions is necessary to obtain a modified polypeptide that is active on a tri-, di- or monoglyceride substrate.

[ 0058 ] In other embodiments, isolated or recombinant polypeptides of the disclosure include: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO : 2 and having one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) subsitutions selected from the group consisting of: N17S, R33T, L64I, E67A, A70T, M119I, A153V, G181C, G202E, K208N, F225L, S238C, I255F, G266S, D270N, and Q277L; (b) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, or 85; (c) a polypeptide comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, or 98% sequence identity to the amino acid sequence set forth in (a) or (b) excluding any change at amino acid residues 17, 33, 64, 67, 70, 119, 153, 181, 202, 208, 225, 238, 255, 266, 270, and 277 of SEQ ID NO: 85 and wherein the polypeptide has lipase activity and improved methanol and/or heat stability compared to a polypeptide of SEQ ID NO: 2; or (d) a polypeptide comprising an amino acid sequence that can be optimally aligned with the sequence of SEQ ID NO:85 (see, e.g., Figure 8) having at least 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 85 while maintain the amino acids at positions 17, 33, 64, 67, 70, 119, 153, 181, 202, 208, 225, 238, 255, 266, 270, and 277 of SEQ ID NO: 85 and wherein the polypeptide has lipase activity and increased methanol and/or thermostability compared to a polypeptide comprising SEQ ID NO : 2. In any of the foregoing polypeptides, the polypeptide has lipase activity and improved tolerace to methanol and/or heat.

[ 0059 ] Similarly, it is understood that the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or non-coding sequence, is a conservative variation of the basic nucleic acid.

[ 0060 ] One of skill in the art will appreciate that many conservative variations of the nucleic acid constructs which are disclosed yield a functionally identical construct. For example, owing to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid.

[ 0061 ] It will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding modified lipase polypeptides of the disclosure may be produced, some of which bear substantial identity to the nucleic acid sequences explicitly disclosed herein. For instance, codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine . Thus, at every position in the nucleic acids of the disclosure where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence .

[ 0062 ] Thus, the disclosure also provides isolated

polynucleotides encoding a polypeptide of the disclosure. In one embodiment, the disclosure provides a novel family of isolated or recombinant polynucleotides referred to herein as "lipase

polynucleotides" or "lipase nucleic acid molecules." Lipase

polynucleotide sequences are characterized by the ability to encode a lipase polypeptide of the disclosure. In general, the disclosure includes any nucleotide sequence that encodes any of the lipase polypeptides described herein.

[ 0063 ] The polynucleotides of the disclosure have a variety of uses in, for example recombinant production (i.e., expression) of the lipase polypeptides of the disclosure and as substrates for further diversity generation, e.g., recombination reactions or mutation reactions to produce new and/or improved lipase homologues, and the like.

[ 0064 ] Lipase polynucleotides, including nucleotide sequences that encode lipase polypeptides and variants thereof, fragments of lipase polypeptides, related fusion proteins, or functional

equivalents thereof, are used in recombinant DNA molecules that direct the expression of the lipase polypeptides in appropriate host cells, such as bacterial cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can also be used to clone and express the lipase polynucleotides. The term "host cell", as used herein, includes any cell type which is susceptible to transformation with a nucleic acid construct. The term

"transformation" means the introduction of a foreign (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by the genetic machinery of the cell. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone." The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

[ 0065 ] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms preferentially use a subset of these codons . The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons (see, e.g., Zhang et al .

(1991) Gene 105:61-72; incorporated by reference herein) . Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called "codon optimization" or "controlling for species codon bias."

[ 0066 ] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al .

(1989) Nucl. Acids Res. 17:477-508; incorporated by reference herein) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E. coli prefer to use UAA as the stop codon (Dalphin et al . (1996) Nucl. Acids Res. 24: 216-218; incorporated by reference herein) . Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No.

6,015,891, and the references cited therein (incorporated herein by reference) .

[ 0067 ] A nucleic acid molecule of the disclosure, e.g., a nucleic acid molecule that encodes a polypeptide of the disclosure are set forth in the accompanying sequence listing.

[ 0068 ] A nucleic acid of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[ 0069 ] In another embodiment, an isolated nucleic acid molecule of the disclosure encoding a polypeptide having lipase activity and methanol and/or thermostability hybridizes under stringent conditions to a nucleic acid molecule consisting of the nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, or 85. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one polynucleotide can anneal to another polynucleotide under defined stringency conditions. Stringency of hybridization is determined, e.g., by (a) the temperature at which hybridization and/or washing is performed, and (b) the ionic strength and polarity (e.g., formamide) of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two polynucleotides contain substantially complementary

sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5 X SSC at 65°C) requires that the sequences exhibit some high degree of complementarity over their entire sequence.

Conditions of intermediate stringency (such as, for example, an aqueous solution of 2 X SSC at 65°C) and low stringency (such as, for example, an aqueous solution of 2 X SSC at 55°C) , require

correspondingly less overall complementarity between the hybridizing sequences (1 X SSC is 0.15 M NaCl, 0.015 M Na citrate). Nucleic acid molecules that hybridize include those which anneal under suitable stringency conditions and which encode polypeptides or enzymes having the same function, such as the ability to catalyze the converstion of a tri-, di- or monoglyceride to a fatty acid. Further, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 30%, 40%, 50%, or 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.

[ 0070 ] Also contemplated are those situations where it is desirable to alter the activity of a parent polypeptide such that the polypeptide has new or increased activity on a particular substrate . It is understood that these amino acid substitutions will generally not constitute "conservative" substitutions. Instead, these

substitutions constitute non-conservative substitutions introduced in to a sequence in order to obtain a new or improved activity. For example, a polypeptide comprising SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, or 85, can serve as a source (parental polypeptide) for further rounds of directed evolution or mutation. These polypeptides may also be referred to as "parent" amino acid sequences because substitutions made to their amino acid sequence give rise to modified lipase polypeptides that can have altered or modified activity as compared to the parent sequence .

[ 0071 ] It is also understood that a modified polypeptide can constitute a "parent" polypeptide from which additional substitutions can be made. Accordingly, a parent polypeptide, and a nucleic acid molecule that encodes a parent polypeptide, includes modified polypeptides and not just "wild-type" sequences. For example, the polypeptide of SEQ ID NO: 85 is a modified polypeptide with respect to SEQ ID NO:2 (i.e., the "parent" polypeptide).

[ 0072 ] Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling et al . (1997) "Approaches to DNA mutagenesis: an overview" Anal Biochem. 254(2) : 157-178; Dale et al . (1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method" Methods Mol . Biol. 57:369-374; Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19:423-462; Botstein &

Shortle (1985) "Strategies and applications of in vitro mutagenesis" Science 229:1193-1201; Carter (1986) "Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds . , Springer Verlag, Berlin) ) ; mutagenesis using uracil containing templates (Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc . Natl. Acad. Sci. USA 82:488-492; Kunkel et al . (1987) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Methods in Enzymol. 154, 367-382; and Bass et al . (1988) "Mutant Trp repressors with new DNA-binding specificities" Science 242:240-245); oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350

(1987) ; Zoller & Smith (1982) "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983) "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors" Methods in Enzymol. 100:468-500; and Zoller & Smith (1987) "Oligonucleotide- directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template" Methods in Enzymol.

154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor et al . (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA" Nucl . Acids Res. 13: 8749- 8764; Taylor et al . (1985) "The rapid generation of oligonucleotide- directed mutations at high frequency using phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787; Nakamaye & Eckstein (1986) "Inhibition of restriction endonuclease Nci I cleavage by

phosphorothioate groups and its application to oligonucleotide- directed mutagenesis" Nucl. Acids Res. 14: 9679-9698; Sayers et al.

(1988) "Y-T Exonucleases in phosphorothioate-based oligonucleotide- directed mutagenesis" Nucl. Acids Res. 16:791-802; and Sayers et al .

(1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide" Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) "The gapped duplex DNA approach to oligonucleotide-directed mutation construction" Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol. "Oligonucleotide-directed construction of mutations via gapped duplex DNA" 154:350-367; Kramer et al. (1988) "Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide- directed construction of mutations" Nucl . Acids Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999) (each of which is

incorporated by reference) .

[ 0073 ] Additional suitable methods include point mismatch repair

(Kramer et al. (1984) "Point Mismatch Repair" Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al . (1985) "Improved oligonucleotide site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using M13 vectors" Methods in Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh &

Henikoff (1986) "Use of oligonucleotides to generate large deletions" Nucl. Acids Res. 14: 5115), restriction-selection and restriction- purification (Wells et al . (1986) "Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin" Phil. Trans. R. Soc . Lond. A 317: 415-423), mutagenesis by total gene synthesis (Nambiar et al . (1984) "Total synthesis and cloning of a gene coding for the ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide- binding protein (transducin) " Nucl. Acids Res. 14: 6361-6372; Wells et al . (1985) "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites" Gene 34:315-323; and Grundstrom et al . (1985) "Oligonucleotide-directed mutagenesis by microscale shot-gun gene synthesis" Nucl. Acids Res. 13: 3305- 3316); double-strand break repair (Mandecki (1986); Arnold (1993) "Protein engineering for unusual environments" Current Opinion in Biotechnology 4:450-455; and "Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli : a method for site- specific mutagenesis" Proc . Natl. Acad. Sci. USA, 83:7177-7181) (each of which is incorporated by reference) . Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods. [ 0074 ] Additional details regarding various diversity generating methods can be found in the following U.S. patents, PCT publications, and EPO publications: U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), "Methods for In vitro Recombination;" U.S. Pat. No. 5,811,238 to Stemmer et al . (Sep. 22, 1998) "Methods for Generating

Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" U.S. Pat. No. 5,830,721 to Stemmer et al . (Nov. 3, 1998), "DNA Mutagenesis by Random Fragmentation and Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) "End- Complementary Polymerase Reaction;" U.S. Pat. No. 5,837,458 to

Minshull, et al . (Nov. 17, 1998), "Methods and Compositions for Cellular and Metabolic Engineering;" WO 95/22625, Stemmer and

Crameri, "Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" WO 97/35966 by Minshull and Stemmer, "Methods and Compositions for Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al . "Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et al . "Antigen Library Immunization;" WO 99/41369 by Punnonen et al . "Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et al . "Optimization of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and Reassembly;" EP 0932670 by Stemmer

"Evolving Cellular DNA Uptake by Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al . , "Modification of Virus Tropism and Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al . , "Human Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al . "Evolution of Whole Cells and Organisms by Recursive Sequence

Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/13487 by Stemmer et al . , "Methods for Optimization of Gene Therapy by Recursive Sequence Shuffling and Selection;" WO 00/00632, "Methods for Generating Highly Diverse Libraries;" WO 00/09679, "Methods for Obtaining in vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences;" WO 98/42832 by Arnold et al . , "Recombination of Polynucleotide Sequences Using Random or Defined Primers;" WO 99/29902 by Arnold et al., "Method for Creating Polynucleotide and Polypeptide Sequences;" WO 98/41653 by Vind, "An in vitro Method for Construction of a DNA Library;" WO 98/41622 by Borchert et al . , "Method for Constructing a Library Using DNA Shuffling;" WO 98/42727 by Pati and Zarling, "Sequence Alterations using Homologous Recombination;" WO 00/18906 by Patten et al . , "Shuffling of Codon-Altered Genes;" WO 00/04190 by del Cardayre et al . "Evolution of Whole Cells and Organisms by Recursive Recombination;" WO 00/42561 by Crameri et al . , "Oligonucleotide Mediated Nucleic Acid Recombination;" WO 00/42559 by Selifonov and Stemmer "Methods of Populating Data Structures for Use in

Evolutionary Simulations;" WO 00/42560 by Selifonov et al . , "Methods for Making Character Strings, Polynucleotides & Polypeptides Having Desired Characteristics;" WO 01/23401 by Welch et al . , "Use of Codon- Varied Oligonucleotide Synthesis for Synthetic Shuffling;" and WO 01/64864 "Single-Stranded Nucleic Acid Template-Mediated

Recombination and Nucleic Acid Fragment Isolation" by Affholter (each of which is incorporated by reference) .

[ 0075 ] Also provided are recombinant constructs comprising one or more of the nucleic acid sequences as described above. The constructs comprise a vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC) , a yeast artificial chromosome

(YAC) , or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences including, for example, a promoter operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

[ 0076 ] Accordingly, in other embodiments, vectors that include a nucleic acid molecule of the disclosure are provided. In other embodiments, host cells transfected with a nucleic acid molecule of the disclosure, or a vector that includes a nucleic acid molecule of the disclosure, are provided. Host cells include eukaryotic cells such as yeast cells, insect cells, or animal cells. Host cells also include procaryotic cells such as bacterial cells.

[ 0077 ] The terms "vector", "vector construct" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a "plasmid", which generally is a self-contained molecule of double- stranded DNA that can readily accept additional (foreign) DNA and which can be readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of

eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech) , pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego,

Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes .

[ 0078 ] The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be

"expressed" by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.

[ 0079 ] Polynucleotides provided herein can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies , adenovirus, adeno-associated viruses, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used.

[ 0080 ] Vectors can be employed to transform an appropriate host to permit the host to express a protein or polypeptide. Examples of appropriate expression hosts include: bacterial cells, such as E. coli, B. subtilis, Streptomyces, and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and

Neurospora crassa; insect cells such as Drosophila and Spodoptera frugiperda; mammalian cells such as CHO, COS, BHK, HEK 293 br Bowes melanoma; or plant cells or explants, etc.

[ 0081 ] In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the lipase

polypeptide. For example, when large quantities of lipase polypeptide or fragments thereof are needed for commercial production or for induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be desirable. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene) , in which the lipase polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J. Biol. Chem. 264: 5503-5509); pET vectors (Novagen, Madison Wis.); and the like.

[ 0082 ] Similarly, in the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used for production of the lipase polypeptides of the invention. For reviews, see Ausubel

(supra) and Grant et al . (1987) Methods in Enzymology 153:516-544

(incorporated herein by reference) .

[ 0083 ] Also provided are engineered host cells that are

transduced (transformed or transfected) with a vector provided herein

(e.g., a cloning vector or an expression vector), as well as the production of polypeptides of the disclosure by recombinant

techniques. The vector may be, for example, a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the lipase gene. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Sambrook, Ausubel and Berger, as well as e.g., Freshney (1994) Culture of Animal Cells: A Manual of Basic Technique, 3rd ed. (Wiley-Liss, New York) and the references cited therein .

[ 0084 ] As previously discussed, general texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, are available to the skilled artisan and include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152, (Academic Press, Inc., San Diego, Calif.) ("Berger"); Sambrook et al . ,

Molecular Cloning--A Laboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds . , Current Protocols, a joint venture between Greene Publishing

Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ("Ausubel"); Innis et al . , eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press Inc. San Diego, Calif.) ("Innis") (each of which is incorporated by reference) .

[ 0085] The invention is illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting .

EXAMPLES

[ 0086] Reagents . All chemicals were of analytical grade or better. 4-nitrophenyl palmitate, and Triton X100 were from Sigma. 1- naphthyl palmitate and Fast Blue B was from MPI Biochemicals . All other solvents were from Fluka. Amano lipase PS (Burkholderia cepacia) was purchased from Sigma and was purified before use using a HiTrapQ column (GE life sciences) and dialyzed into 20 mM Tris-Cl pH 7.5, 0.1 M NaCl . Primers for cloning and mutagenesis were ordered from Valuegene . Canola oil used for transesterification was from the local market. [ 0087 ] Construction of Mutants by Site-Directed Mutagenesis.

Site-Directed mutagenesis was performed using the Quickchange Site Directed Mutagenesis Kit (Stratagene) according to the manufacturer's directions. For generation of double mutants, a modified megaprimer method was used. Briefly, in an initial PCR, a forward and reverse mutagenic primer, each containing a different mutation, was used to amplify a short segment of the PML gene using Taq Hot-Start Supermix (Denville) to generate a megaprimer. In a second PCR reaction, 5 L of the initial PCR was mixed with 30 ng template, 0.2 μΜ dNTP, Pfu HotStart II Reaction Buffer, and 2.5 U of Pfu HotStart Fusion II DNA Polymerase (Stratagene) in a 50 L reaction. The reaction was cycled as for the Quickchange reaction. The resulting PCR product was digested with Dpnl and used to transform BL21Gold (DE3) or XLlOGold directly .

[ 0088 ] Construction of PML Mutant Library by error-prone PCR. The wild-type lipase gene from Proteus mirabilis was cloned into a pET28 vector as previously described and used as template for error prone PCR. Random mutagenesis was performed using the Genemorph II Kit (Stratagene) according to the manufacturer's instructions. Primers which flank the 5'NdeI and 3'EagI restriction sites were used for amplification. An appropriate amount of DNA template was used to generate between 1 and 5 mutations per 1 kb. Briefly, 0.1 ng template was mixed with 0.2 mM each dNTP, 0.2 μΜ each primer, IX Mutazyme II Buffer, and 2.5 U Mutazyme II in 50 L . The PCR reaction incubated at 95 °C for 2 minutes followed by 45 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, with a final extension at 72 °C for 10 minutes. The resulting PCR product was purified using a QIAquick spin column and digested overnight with Ndel and Eagl (NEB) at 37 °C. The digested product was gel purified and ligated into pET28 that had been digested with the same enzymes using T4 DNA ligase (NEB) for 16 hours at 16 °C. The resulting library was directly transformed into chemically competent BL21Gold (DE3) and plated on LB-agar containing 50 g/mL kanamycin for analysis and screening.

[ 0089 ] Library Screening for MeOH Tolerance. Screening for improved methanol tolerance was accomplished by a colony lift screening protocol with a PML mutant library expressed in

BL21Gold (DE3) . Following overnight incubation at 37 °C, transformants (~600/plate) were lifted onto sterile filter circles (Whatman 410) and placed colony-up on a LB-agar plate containing 50 g/mL kanamycin and 1 mM IPTG for 2-3 hours at 18 °C to induce protein expression. The filter was then immersed in lysis solution (50 mM Tris-Cl pH 7.5, 0.1 M NaCl, 0.1% Triton X100, 1 mg/mL lysozyme) at 25 °C for 1 hour. Lysis solution was decanted and replaced by methanol solution containing 0.1% Triton X100 and incubated at a selected temperature for a desired amount of time. After incubation, the methanol solution was decanted and the filters were developed by overlaying with 1 mM 1-naphthyl palmitate, 3 mM Fast Blue B, 0.5% Triton X100 dispersed in 0.5% agar. After 10 minutes, mutants displaying residual activity were identified by formation of a purple color and the corresponding colony isolated from the master plate for validation and further characterization .

[ 0090 ] For validation, positives from the filter screen were grown in 10 mL LB containing 50 μg/mL kanamycin to Οϋεοο of 0.6 and protein expression induced with 0.5 mM IPTG for 16 hours at 18 °C. The cells were pelleted, resuspended in buffer (50 mM Tris-Cl pH 7.5, 0.1 M NaCl, 5 mM imidazole), lysed by sonication (5 x 30 s pulse) and clarified by centrifugation (13K rpm x 30 min; Sorval SS-34 rotor) . To validate, 50 L of the supernatant was incubated with 70% MeOH for 1 hour and then diluted to 10% MeOH prior to being assayed with 1 mM p-nitrophenyl palmitate (pNPP) . Residual activity was defined as the activity compared to a sample incubated at 10% MeOH for 1 hour.

Mutations were confirmed by sequencing (Genewiz) and the best performing mutants were combined by site-directed mutagenesis and reassayed for improved MeOH tolerance. The best performing combined mutants were then used as parents for subsequent rounds of directed evolution .

[ 0091 ] Expression and Purification of WT and Mutant PMLs . Over- expression of wild-type and mutant PML was done in E. coli

BL21Gold (DE3) (Agilent) . Single transformants were transferred to 2L of Luria-Burtani (LB) media containing kanamycin at 37 °C to Οϋεοο of 0.6. Protein expression was induced with 0.5 mM IPTG at 16 °C for 16 hours. Cells were harvested by centrifugation and purified by Ni-NTA chromatography (Qiagen) as described previously. Wild-type and mutant PMLs were then dialyzed into 20 mM Tris-Cl pH 7.5 containing 100 mM NaCl and flash frozen as droplets in liquid N2 prior to storage at -80 °C.

[ 0092 ] Enzyme Kinetics and Characterization. Kinetic assays were carried out in 96 well microtiter plates (Grenier Bio-One) with purified protein. For kinetic analysis a stock solution of 86.13 mM pNPP was prepared in 1:1 Acetonitrile : Triton X100. The initial rate of conversion of pNPP to p-nitrophenol was monitored at 405 nm over the first minute using a plate reader (SpectraMax M5, Molecular Devices) . For temperature or methanol incubation studies, the residual activity was assayed using 1 mM pNPP in 50 mM phosphate pH 7.0. For temperature inactivation, 60 L of 150 nM enzyme was incubated in 50 mM phosphate pH 7.0 in thin walled PCR tubes at various temperatures for 1 hour or with time at 50 °C using a PCR cycler (Mastercycler ProS, Eppendorf) . Activity was normalized to activity at 25 °C. For methanol tolerance, 1.5 μΜ enzyme was

incubated with 50 mM phosphate pH 7.0 containing methanol. After 2 hours, the enzyme was diluted 1:10 so that the final methanol concentration was 10 % and the final enzyme concentration was 150 nM. For inactivation by methanol over time, the enzyme was incubated in 50% methanol at 25 °C. At various time points, 10 L aliquots were diluted 1:10 with 90 L 5.5 % methanol (10% final) and assayed for residual activity compared to incubation with 10% methanol as described above.

[ 0093 ] Immobilization and Transesterification . Purified B.

cepacia, PML, and mutant lipases (Dieselzymes) were covalently immobilized on hydrophobic Immobead 350 oxirane functionalized beads

(ChiralVision) prior to use. Beads (0.25 g) were washed once with 10 mL methanol followed by two washes with 10 mL 0.1 M phosphate pH 7.0. The buffer was decanted and 1 mL enzyme at 0.2 mg/mL in 20 mM tris-Cl pH 7.5, 0.1 M NaCl was added. Immobilization was allowed to proceed for 16-20 hours at 25 °C. All 3 enzymes studied were immobilized to a similar degree (>95%) as monitored by OD₂80nm and by residual activity remaining in the supernatant.

[ 0094 ] After immobilization, the buffer was decanted and the beads were used for transesterification without further modification. For the synthesis reaction, 0.625 mL of 50% methanol (1:1

methanol : 0.1 M phosphate pH 7.0) was added to the beads followed by 1.5 mL refined canola oil. The solution was gently vortexed and then placed on a shaker at 200 rpm at 25 °C. An initial reaction was performed with 0.1 g beads to establish the transesterification rate for each construct. For the recycling experiment, an appropriate amount of beads (0.25 g, 0.25 g, and 0.08 g of wild-type PML,

Dieselzyme 4, and BCL respectively) were added such that -5-10% conversion was reached in the first hour. After each 20 hour cycle, 10 L aliquots of the oil layer were taken for analysis by GC

(below) . To monitor the effect of reuse on initial rate, 10 L were taken at 2 and 4 hours during cycles 1, 4, and 7. For reuse, the beads were recovered by filtration, washed with 5 mL buffer followed by 5 mL hexanes and then allowed to dry before adding fresh methanol, buffer, and oil.

[ 0095 ] Quantification of Fatty Acid Methyl Esters. The extent of transesterification was monitored by gas chromatography (GC) . 10 L samples were diluted with 1 mL hexanes spiked with 0.5 mg/mL methyl heptadecanoate (internal standard) . Samples were analyzed on an Agilent 5890 Series II GC with flame ionized detector using an HP- INNOWax column (0.25 mm x 30 m, Agilent) . The oven temperature was kept at 200 °C for 3 min and then raised to 230 at 5 °C/min then to 250 at 20 °C/min and held at 250 °C for 9 min. The injector and detector temperatures were kept at 230 and 330 °C respectively. The percent conversion was determined by comparison to a biodiesel sample prepared from canola oil using a large excess of free cepacia lipase at a 5:1 MeOH:oil ratio in the presence of 5% water as described previously .

[ 0096 ] Crystallization, Structure Determination, and Refinement of Mutant PML. Crystallization trials for Dieselzyme 4 were performed using purified His-tagged lipase with no additives. Drops were generated by mixing 2 μL purified protein at 9 mg/mL with 2 μL well solution. Large crystals of the mutant PML formed in many conditions between one day and two weeks. One condition, Qiagen PACT condition #38 (IX MMT pH 5, 20% PEG 1500), was optimized and gave large crystals (0.2x0.2x0.2 mm) within one week. Prior to data collection, crystals were soaked in crystallization well solution plus 15% glycerol and flash frozen in liquid nitrogen. Data were collected in house on a Rigaku FRE+ x-ray generator equipped with an ADSC Quantum 4 CCD detector at 100 K. Diffraction images were indexed, integrated, and scaled with Denzo and Scalepack. Initial phases were determined by Molecular Replacement using PHASER in CCP4i. The wild-type PML was used as the search model. The resulting model was further refined via iterative rounds of model building and refinement in COOT and Refmac5 over the resolution range 50-1.8 A.

[0097] Dieselzyme 1: Generation of Disulfide Mutant increases stability. The introduction of disulfide bonds is a common strategy to improve enzyme stability that has successfully been applied to other lipases. Based on sequence analysis, it was hypothized that one could stabilize the PML by the introduction of a disulfide bond.

Homologous lipases from Pseudomonas aeruginosa and Burkholderia cepacia contain a single disulfide bond between residues 181 and 238

(PML numbering) that is not conserved in PML. The PML wild-type structure showing the location of the corresponding disulfide bond in B. cepacia (10IL) can be seen in Figure 2A. Due to the structural conservation of this region and the proximity of G181 and S238 in PML a disulfide bond was introduced between residues G181 and S238 by mutation to cysteines (FIGURE 2A) . This mutant is referred to as Dieselzyme 1.

[0098] The effect of the introduced disulfide bond on resistance to thermal inactivation was determined by monitoring the residual lipase activity after heat treatment. Samples were incubated for 1 hour between 37 and 60 °C and the residual activity measured. As shown in Figure 2B, the introduction of a single disulfide in

Dieselzyme 1 significantly stabilized PML, increasing the half- inactivation temperature (IT1_/2) from 37 °C for the wild-type enzyme to 48 °C for the Dieselzyme 1. Also, when incubating at a constant 50 °C, the half life increased from less than 15 min for wild-type to -75 min for Dieselzyme 1. Interestingly, Dieselzyme 1 displays the same temperature inactivation profile as the native Burkholderia cepacia lipase (BCL) which contains a single disulfide in the same position (Figure 2B) .

[0099] While thermostability is an important property that contributes to the industrial usefulness of an enzyme, a more thermostable enzyme does not necessarily mean that an enzyme will be more tolerant to organic solvents, especially relatively polar, water miscible solvents such as methanol and ethanol that are used in biodiesel synthesis. Indeed, in work with Pseudomonas fragi lipase, increasing thermostability did not affect solvent tolerance.

Moreover, many thermostable lipases such as the Thermomyces

lanuginosus lipase display only modest alcohol tolerance. The effect of methanol on wild-type PML and Dieselzyme 1 was monitored by measuring the residual activity after incubation with various concentrations of methanol for 2 hours at 25 °C. Figure 2C,D shows that introduction of a disulfide bond in PML does not have an effect on the methanol tolerance of PML as the methanol inactivation profiles for wild-type PML and Dieselzyme 1 are identical. Thus, consistent with earlier work, it appears that the mechanism of methanol inactivation is different from heat inactivation. Directed evolution was employed to improve the methanol tolerance Dieselzyme- 1.

[00100] Dieselzymes 2-4: Directed Evolution. To screen for mutants with improved methanol and heat tolerance, a rapid colony assay for stability was developed (Figure 3) . Following error-prone PCR, mutant library colonies were then lifted onto filter paper and protein expression induced by placing the colonies face up on LB-agar containing 1 mM IPTG for 3 hours. The lipase variants expressed in each colony were then tested for their resistance to inactivation by incubating the filters in heat and methanol. After removal of the methanolic incubation solution, mutants that retained significant residual activity could be identified by overlaying with a solution of 1-naphthyl palmitate and Fast Blue B dye suspended in agar, leading to the formation of a dark purple/brown colony when active lipase is present.

[00101] To screen for improved methanol tolerance, the stable disulfide mutant Dieselzyme 1 was used as the parent for error-prone PCR. In the first round, the mutant library was incubated with 50% methanol at 42 °C for 45 minutes prior to screening for residual activity. In the second round, the mutant library was incubated with 65% methanol at 42 °C for 45 minutes. Finally, the third round of screening was performed in 65% methanol at 47 °C for 90 minutes.

During each round of screening, only the mutant clones displaying residual activity within the first ten minutes were picked and confirmed in a second colony screen as above. Typically, 10-15 mutants out of -20000 were selected for further validation during each round of screening.

[ 00102 ] The mutant lipases positive for increased methanol resistance after each round were purified and tested for their tolerance to methanol by measuring residual activity after a 16 hr incubation in 70% methanol at 25 °C. Mutations from improved mutants identified in each round were combined iteratively via site-directed mutagenesis. The mutant proteins were then purified and tested for increased methanol tolerance. The best recombinant from each round was then used as the parent for the next round of error-prone PCR. After introduction of the disulfide bond (Dieselzyme 1) and 3 rounds of random mutagenesis with site-directed recombination (Dieselzymes 2-4), a mutant (Dieselzyme 4) with 13 amino acid changes (Table 1) was identified that retained more than 80% of its residual activity after incubation with 70% methanol for 16 hours and displayed further improved thermostability compared to the disulfide mutant Dieselzyme 1.

[ 00103] Characterization of Thermostability and Alcohol Tolerance of Evolved PML Mutants. To evaluate the full extent of stabilization after random mutagenesis, the residual activity of wild-type PML was compared with each Dieselzyme variant as a function of temperature or methanol concentration as shown in Figures 4 and 5 respectively. The thermal inactivation profile for the disulfide bond mutant Dieselzyme 1 and Dieselzyme 2 are identical. However, in Dieselzyme 3 and

Dieselzyme 4, the added mutations have a beneficial effect on thermostability with an apparent T1/2 after 1 hr of 55 °C for both Dieselzyme 3 and Dieselzyme 4, an increase of 17 °C compared to wild- type and ~5 °C higher than Dieselzyme 1 (Figure IB, 4A) . In

addition, when the mutant lipases were incubated at a constant 50 °C, the final mutant Dieselzyme 4 also showed a further improvement over Dieselzyme 3 with an inactivation half-life of ~7 hours (Figure 4B) which is more than a 30 fold improvement over wild-type (less than 15 min) . Dieselzyme 3 and Dieselzyme 4 are also more resistant to thermal inactivation than the lipase from B. cepacia described above.

[ 00104 ] The methanol tolerance of wild-type and mutant PMLs as a function of methanol concentration is shown in Figure 5A. Due to activation of PML in low concentrations of methanol, the residual activity was normalized to activity remaining after incubation with 10% methanol. Similar activation of related lipases with short-chain alcohols has been demonstrated. When the wild-type or Dieselzyme 1 is incubated for two hours at increasing methanol concentrations, both enzymes retain 50% activity in 55% methanol and are completely inactivated at 70% methanol. In contrast, Dieselzyme 2 retained -20% residual activity after incubation with 80% methanol while the

Dieselzyme 3 and Dieselzyme 4 retain greater than 70% of their activity after incubation in 70% methanol.

[00105] While the absolute methanol tolerance of PML was improved during a relatively short incubation, the actual goal was to develop a mutant lipase that had better long-term resistance to the presence of methanol. In order to determine if the mutant lipases were also more resistant to methanol inactivation over a long period of time, wild-type or mutant PML was incubated for 24 hours in the presence of 50% methanol. Figure 5B shows the residual activity of wild-type and mutant PML from the different rounds of screening after treatment with 50% methanol as a function of time. The long-term resistance was greatly improved by the screening regimen, with Dieselzyme 4 retaining roughly 90% activity after incubation with 50% methanol for 24 hours. B. cepacia lipase that is currently used industrially for biodiesel synthesis shows a comparable level of tolerance under these conditions. As shown in Fig. 5C, however, when the concentration of methanol was raised to 70%, Dieselzyme 1 retains essentially full activity after 24 hrs, but the B. cepacia lipase retains only -60%.

[00106] Other Alcohols and Water Miscible Solvents. To test whether the improvement in methanol tolerance also rendered

Dieselzyme 4 more resistant to other water-miscible organic solvents, the enzymes was incubated in the presence of a variety of organic solvents at a concentration of 70% for 24 hours, and then assayed for residual hydrolysis activity (FIGURE 5C) . In general, Dieselzyme 4 retained considerably more activity than the wild-type after incubation with organic solvents, with the exception of DMSO . DMSO surprisingly stimulates the wild-type PML enzyme, but has little effect on Dieselzyme 4. [00107] The organic solvent tolerance of Dieselzyme 4 was compared to the tolerance of the B. cepacia lipase. For the alcohols most commonly used in biodiesel synthesis, methanol and ethanol,

Dieselzyme 4 is more tolerant. The B. cepacia lipase is more tolerant to propanol, acetonitrile and acetone, however, indicating that organic solvent tolerance is a complex property.

[00108] Transesterifcation and Recycling of Immobilized PML. The disclosure provides a stable lipase that is less prone to methanol induced inactivation during biodiesel synthesis. The ability of Dieselzyme 4 to remain active in the transesterification reaction was tested for many cycles over a long period of time. To monitor both transesterification ability and resistance to methanol induced inactivation during multiple rounds of synthesis, wild-type,

Dieselzyme 4, and the industrial enzyme BCL were covalently

immobilized onto hydrophobic oxirane functionalized beads. To monitor biodiesel synthesis, beads were added to a mixture of 0.625 ml of 50% methanol and 1.5 ml of canola oil. This mixture provides a -5:1 molar ratio of methanol to triacylglycerol oil or a -1.6 molar ratio of methanol per ester bond. The final water content was -30% (w/w) of the oil. To test the response of the enzymes to prolonged exposure to methanol, a low amount immobilized enzyme beads were added such that there was less than 10% conversion in the first hour. For each cycle the progress of the reaction was monitored after 20 hours. For the first, fourth and seventh cycles, the reaction progress was also monitored at 2, 4 and 6 hours to observe initial rates. To restart the reaction at each cycle, the beads were separated by filtration and washed prior to reuse in a subsequent round of biodiesel synthesis. A water wash was performed to remove glycerol and residual methanol followed by a wash with hexanes to remove residual oil and fatty acid methyl esters.

[00109] The results of multiple rounds of transesterification can be seen in Figure 6. During the first cycle the initial rate of biodiesel synthesis was linear and showed a similar rate of

conversion for all three immobilized enzymes, with 24.7%, 20.6%, and 36.5% conversion to methyl esters for WT, Dieselzyme 4, and BCL after 4 hours. The rate of synthesis eventually decreased for both WT and BCL, however, such that only 47.7% and 56.4% total conversion was seen after 20 hours. In contrast, Dieselzyme 4, continued at a high rate of synthesis, converting -76% of the canola oil to biodiesel within 20 hours. Thus, even though there was higher initial BCL activity, Dieselzyme 4 was able to convert more oil over the longer incubation .

[00110] The improved methanol tolerance of Dieselzyme 4 becomes even more apparent when the immobilized enzymes are reused in a second cycle. After a single reuse, the wild-type PML lost nearly all of its ability to catalyze transesterification with less than 15% of the canola oil being converted to biodiesel in the second round. Similarly, BCL only converted 30% in the second round. In contrast, Dieselzyme 4 retained complete activity in the second round.

Moreover, the rate of inactivation of Dieselzyme 4 was significantly slower than that of BCL as Dieselzyme 4 retained nearly the same amount of activity after round 4 compared to round 1 and still converted 50% of canola oil to biodiesel after the fifth cycle

(compared to only 18% for BCL after cycle 5) . These results show that not only is Dieselzyme 4 active for transesterifcation, but

Dieselzyme 4 is significantly more resistant to inactivation during biodiesel synthesis compared to wild-type PML and even outperforms BCL .

[00111] Structural Basis for Evolved Methanol Tolerance. The structure of Dieselzyme 4 was determined by x-ray crystallography to examine the effects of the mutations on the PML structure. This is the first structure of an enzyme that has been evolved for increased organic solvent tolerance. An analysis of the crystal structure of Dieselzyme 4 suggests that mutations in 2 key areas, (Region 1 and Region 2) may be important for the improvement in methanol tolerance (Figure 7A and 7B) .

[00112] Region 1 comprises the mutations R33T, L64I, and A70T that cluster on or near helices al and oc2 at opposite end of the protein from the lid region (Figure 7B and 7C) . Well defined electron density was seen for all three of the new side chains. Mutation of L to I at position 64 provides more interactions with side-chains F40 and F60, possibly improving packing to stabilize helix oc2. The A70T mutation, which arose in the first round of error-prone PCR

(Dieselzyme 2), shows that the hydroxyl from the threonine forms a new H-bond interaction with T68 and the backbone carbonyl of K72. Similarly, the R33T mutation that arose after the final round of error-prone PCR (see Dieselzyme 4) forms new H-bond interactions between the threonine hydroxyl and the backbone carbonyls from A29 and D30. In organic solvents such as methanol, it is possible that H- bond interactions would be strengthened. Thus, the formation of new H-bonds might partially explain the increase in methanol tolerance as a result of the R33T and A70T mutations.

[ 00113 ] Region 2 mutations (Figure 7E and 7F) cluster near the Ca²⁺ binding site, and cause an unexpected remodeling of the loop between residues 200 and 208 that possibly stabilizes the Ca²⁺ binding site and provides stabilizing interactions between the loop lid helix a8. While the density of the remodeled loop is not as well defined as the rest of the protein, there is sufficient electron density to provide a rational for the observed improvement in methanol tolerance.

Mutations in the loop consist of G202E, K208N, and G266S and are located close to the Ca²⁺ binding site essential for lipase stability and activity. The most striking change is a direct result of the G202E mutation. The introduced Glu side chain at position 208 is positioned ~4 A from R220 in an orientation that could form a new salt-bridge. In order to make this potential new salt-bridge, the helical region between residues 203 and 207 seen in wild-type PML partially unravels and may increase flexibility of the loop region to allow new interactions to occur. For instance, the increased

flexibility due to unraveling of the helix between 203 and 207 may allow E207 to move and form a new salt-bridge with R220 and conserved H216. However, the electron density surrounding R220 is split, suggesting that R220 is in at least two conformations: one that can interact with E207 and one that potentially interacts with the introduced G202E mutation (Figure 7F) . Additionally, while the K208N mutation is well defined and does not alter the backbone or side chain conformation at position 208, it is possible that removal of the charged Lys also increases the flexibility of the loop region, facilitating the new interactions made by the G202E mutation and E207. It has been reported for CALB lipase, phospholipase Al, and metalloprotease , that the introduction of polar residues can lead to dramatic increases in solvent tolerance. However, in none of these examples is the change as dramatic as that seen between wild-type PML and Dieselzyme 4.

[ 00114 ] Interestingly, in addition to the polar residue insertion and loop remodeling described above, the G266S mutation forms another new polar interaction, albeit with a crystallographic water and the side chain N210 that directly coordinates the Ca²⁺. Previous

experiments with the homologous lipases from B. glumae have indicated that the bound Ca²⁺ plays an important role in lipase stability.

Therefore, the introduction of a new polar interaction from G266S may help further stabilize the bound Ca²⁺ in Dieselzyme 4. Together, the overall effect of polar mutations in Region 2 is to provide

stabilizing interactions with the lid helix a8 (through R220 and H216) and to potentially stabilize the Ca²⁺ binding site.

[ 00115 ] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS

1. An isolated or recombinant lipase polypeptide comprising a sequence that is at least 80% identical to SEQ ID NO:2 and having at least one or more mutations at a residue selected from the group consisting of R33, L64, A70, G181, G202, K208, S238, G266 and any combination thereof and wherein the polypeptide has lipase activity and improved methanol tolerance and/or thermostability compared to a wild-type lipase of SEQ ID NO: 2.

2. The isolated or recombinant lipase polypeptide of claim 1, wherein the polypeptide has a mutation at G181 and S238 and has improved thermostability compared to a polypeptide comprising SEQ ID NO: 2.

3. The isolated or recombinant lipase polypeptide of claim 2, wherein the mutation at G181 is G181C.

4. The isolated or recombinant lipase polypeptide of claim 2, wherein the mutation at S238 is S238C.

5. The isolated or recombinant lipase polypeptide of any of the foregoing claims wherein the polypeptide is at least 80% identical to SEQ ID NO: 15 and having C181 and C238.

6. The isolated or recombinant lipase polypeptide of any of the foregoing claims wherein the polypeptide is at least 85% identical to SEQ ID NO: 15 and having C181 and C238.

7. The isolated or recombinant lipase polypeptide of any of the foregoing claims wherein the polypeptide is at least 95% identical to SEQ ID NO: 15 and having C181 and C238.

8. The isolated or recombinant lipase polypeptide of any of the foregoing claims wherein the polypeptide comprises SEQ ID NO: 15.

9. The isolated or recombinant lipase polypeptide of any of claim 2-8, wherein the polypeptide further comprises a mutation at a residue selected from the group consisting of R33, L64, A70, G202, K208, G266 or any combination thereof and wherein the lipase has improved methanol tolerance compared to a polypeptide of SEQ ID NO: 2.

10. The isolated or recombinant lipase polypeptide of claim 9, wherein the polypeptide further comprises a mutation selected from the group consisting of R33T, L64I, A70T, G202E, K208N, G266S and any combination thereof.

11. The isolated or recombinant lipase polypeptide of claim 10, wherein the polypeptide comprises the mutations R33T, L64I, A70T, G202E or G202D, K208N, and G266S.

12. The isolated or recombinant lipase polypeptide of any of the foregoing claims further comprising a mutation at a residue selected from the group consisting of L13, N17, F40, V61, E67, A70, S95, R117, M119, T138, S141, S144, G145, D149, A153, G206, E207, N223, F225, G243, L245, 1255, Y267, D270, V272, Q277 and any combination thereof .

13. The isolated or recombinant lipase polypeptide of claim 12, wherein the polypeptide has mutations selected from the group consisting of:

(a) F225L and Q277L;

(b) N17S, M119I, F225L, D270N and Q277L; and

(c) N17S, E67A, M119I, A153V, F225L, I255F, D270N and Q277L.

14. The isolated or recombinant lipase polypeptide of claim 13, wherein the polypeptide is at least 80% identical to SEQ ID NO: 2 and has mutations selected from the group consisting of:

(a) L64I, A70T, G181C, K208N, F225L, S238C and Q277L;

(b) N17S, L64I, A70T, M119I, G181C, G202E, K208N, F225L, S238C, G266S, D270N and Q277L; and

(c) N17S, R33T, L64I, E67A, A70T, M119I, A153V, G181C, G202E, K208N, F225L, S238C, I255F, G266S, D270N and Q277L.

15. The isolated or recombinant lipase polypeptide of claim 14, wherein the polypeptide comprises a sequence as set forth in SEQ ID NO: 47.

16. The isolated or recombinant lipase polypeptide of claim 14, wherein the polypeptide comprises a sequence as set forth in SEQ ID NO: 65.

17. The isolated or recombinant lipase polypeptide of claim 14, wherein the polypeptide comprises a sequence as set forth in SEQ ID NO: 85.

18. The isolated or recombinant lipase polypeptide of claim 1, having a a sequence selected from the group consisting of: SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, or 85.

19. The isolated or recombinant lipase polypeptide of any of the foregoing claims further comprising from 1-20 conservative amino acid substitutions .

20. An isolated or recombinant polynucleotide encoding any of the foregoing polypeptides .

21. The isolated or recombinant polynucleotide of claim 20, wherein the polynucleotide is selected from the group consisting of SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, and 84.

22. A vector comprising the polynucleotide of claim 20 or 21.

23. The vector of claim 22, which is an expression vector.

24. A host cell transfected with a polynucleotide of claim 20 or 21.

25. A host cell transfected with a vector of claim 22 or 23.

26. The host cell of claim 25, wherein the cell is procaryotic or eukaryotic cell.

27. A method of producing a fatty acid from a tri-, di- or mono- glyceride comprising contacting the tri-, di- or mono-glyceride with a polypeptide of any of claims 1-19 under conditions wherein a fatty acid methyl ester is produced.

28. A method of producing a fatty acid from a tri-, di- or mono- glyceride comprising contacting the tri-, di- or mono-glyceride with a host cell of any one of claims 24-26 under conditions wherein a fatty acid methyl ester is produced.