Acid-resistant lipase

Abstract

The present application provides novel polypeptides with lipase activity, which comprise the amino acid sequences shown in SEQ ID NO: 4 or 6, or sequences obtained by substituting, deleting or adding at least one amino acid to the above sequences. The present application also provides polynucleotides encoding the polypeptides, expression vectors containing the above polynucleotides, and host cells. In addition, the present application also relates to the uses of the polypeptides with lipase activity described above.

Description

Field of the Invention

[0001] The present application belongs to the field of enzyme engineering, and specifically relates to polypeptides having lipase activity, nucleic acids encoding the polypeptides, and expression vectors and host cells comprising the nucleic acids encoding the polypeptides. The present application also relates to methods for screening the polypeptides and their uses. Background of the Invention

[0002] Lipase is triacylglycerol acyl hydrolase, which catalyzes the hydrolysis of natural substrate oils to produce fatty acids, glycerol, and mono- or di-glycerides. It is widely used in oil processing, food, medicine, daily chemical and other industries, and is one of the important industrial enzyme preparations.

[0003] Lipases catalyze the hydrolysis of glycerol ester bonds in oils and fats, as well as reactions such as transesterification, esterification, alcoholysis, and acidolysis. Lipases derived from microorganisms are widely used in industrial production due to their diverse activities, ease of cultivation, and high yields. Currently, approximately 2% of microorganisms are known to produce lipases, and these lipase-producing microorganisms belong to at least 65 genera, including 28 bacterial genera, 4 actinomycete genera, 10 yeast genera, and 23 other fungal genera. Thermomyces lanuginosus, a widely distributed fungus with a high growth temperature ceiling, produces the thermostable, industrially valuable lipase, Thermomyces lanuginosus lipase (TL). TL can be used to methylate fatty acids to produce biodiesel. Because the fatty acid substrates used in the reaction are often byproducts of oil refining and contain large amounts of inorganic acids, acid-resistant lipases are more favorable for fatty acid methylation. SUMMARY OF THE INVENTION

[0004] In a first aspect, a polypeptide having lipase activity is provided, comprising or consisting of a sequence selected from the following:

[0005] (a) the amino acid sequence shown in SEQ ID NO: 4 or 6, and

[0006] (b) A sequence obtained by substituting, deleting or adding at least one amino acid from the sequence described in (a), wherein the polypeptide variant obtained by (b) still retains lipase activity.

[0007] In one embodiment, the polypeptide of the present application comprises the amino acid sequence shown in SEQ ID NO: 4 or 6. In a preferred embodiment, the polypeptide consists of the amino acid sequence shown in either SEQ ID NO: 4 or 6. In a more preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO: 6.

[0008] Herein, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is named G2-4 polypeptide, and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 is named G2-4-27R polypeptide.

[0009] In a second aspect, a polynucleotide encoding the polypeptide of the first aspect is provided, which comprises or consists of a sequence selected from the following:

[0010] (a) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 or 6, or a sequence comprising at least one amino acid substitution, deletion or addition in SEQ ID NO: 4 or 6; and

[0011] (b) A nucleotide sequence that hybridizes to the nucleotide sequence in a) under stringent conditions.

[0012] In one embodiment, the polynucleotide of the present application comprises the nucleotide sequence shown in SEQ ID NO: 3 or 5. In a preferred embodiment, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO: 3 or 5. In a more preferred embodiment, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO: 5.

[0013] In a third aspect, an expression vector is provided, which comprises at least one polynucleotide according to the second aspect.

[0014] In certain embodiments, the expression vector of the present application further comprises a regulatory sequence for regulating the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence. In a specific embodiment, the expression vector is a plasmid vector, such as a pAO815 plasmid.

[0015] In a fourth aspect, a host cell comprising the polynucleotide of the second aspect or the expression vector of the third aspect is provided.

[0016] In some embodiments, the host cell is E. coli, or a yeast such as Pichia pastoris.

[0017] In a fifth aspect, there is provided use of the polypeptide described in the first aspect for fatty acid methyl esterification or for preparing biodiesel.

[0018] In a sixth aspect, a method for preparing biodiesel is provided, comprising contacting the polypeptide of the first aspect with a fat feedstock and a lower alcohol. In some embodiments, the lower alcohol is selected from methanol, ethanol, propanol, butanol, and pentanol, or any combination thereof.

[0019] In a seventh aspect, a method for screening the polypeptide of the present application is provided, comprising:

[0020] 1) mutating the nucleotide sequence shown in SEQ ID NO: 1 to obtain a mutant sequence,

[0021] 2) The mutant sequence obtained in step 1) is cloned into an expression vector and then transformed or transduced into a suitable host cell,

[0022] 3) culturing the host cells in step 2), recovering the recombinant expression vector, and then transforming it into a yeast strain for cultivation, and

[0023] 4) Screening for mutant polypeptides having higher enzyme activity and / or acid resistance than the polypeptide shown in SEQ ID NO: 2.

[0024] In addition, the present application also provides the use of the nucleic acid sequence shown in SEQ ID NO: 1 for screening the polypeptide disclosed herein.

[0025] The polypeptide of the present application has good pH stability and strong acid resistance, and can be used for the fatty acid methyl esterification reaction of high acid value oil raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The stability of TL, G2-4 and G2-4-27R at 45°C was shown.

[0027] Figure 2 The stability of TL, G2-4 and G2-4-27R at 45°C and pH 4.2 was shown.

[0028] Figure 3 The results of methyl esterification of TL, G2-4 and G2-4-27R are shown.

[0029] Brief description of sequence

[0030] SEQ ID NO: 1: nucleic acid sequence encoding TL;

[0031] SEQ ID NO: 2: amino acid sequence of TL;

[0032] SEQ ID NO: 3: nucleic acid sequence encoding G2-4;

[0033] SEQ ID NO: 4: amino acid sequence of G2-4;

[0034] SEQ ID NO: 5: nucleic acid sequence encoding G2-4-27R;

[0035] SEQ ID NO:6: amino acid sequence of G2-4-27R;

[0036] SEQ ID NO:7: Amplification primer TL-1;

[0037] SEQ ID NO: 8: Amplification primer TL-2;

[0038] SEQ ID NO:9: Amplification primers AOX1-5;

[0039] SEQ ID NO: 10: Amplification primers AOX1-3. DETAILED DESCRIPTION

[0040] The polypeptide of this application

[0041] The present application provides a polypeptide having lipase activity, which comprises or consists of a sequence selected from the following:

[0042] (a) the amino acid sequence shown in SEQ ID NO: 4 or 6, and

[0043] (b) A sequence obtained by substituting, deleting or adding at least one amino acid from the sequence described in (a), wherein the polypeptide variant obtained by (b) still retains lipase activity.

[0044] In certain embodiments, the number of the above amino acid substitutions, deletions or additions is 1-30, preferably 1-20, more preferably 1-10, wherein the obtained polypeptide variant substantially retains the lipase activity of the unchanged protein.

[0045] In a preferred embodiment, the polypeptide variants differ from the amino acid sequence of SEQ ID NO: 4 or 6 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In a more preferred embodiment, the polypeptide variants differ from the amino acid sequence of SEQ ID NO: 4 or 6 by about 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions.

[0046] In some embodiments, the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 4 or 6. In a preferred embodiment, the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 6. Herein, the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 is designated as G2-4, and the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 is designated as G2-4-27R.

[0047] In some embodiments, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are made in the amino acid sequence of the G2-4 or G2-4-27R polypeptide, and the polypeptide after conservative substitutions substantially retains the lipase activity of the original polypeptide.

[0048] In other embodiments, the G2-4 or G2-4-27R polypeptide can have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids deleted from the C-terminus or N-terminus and still have lipase activity.

[0049] In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added to the C-terminal or N-terminal region of the G2-4 or G2-4-27R polypeptide, and the resulting polypeptide variant still has lipase catalytic activity.

[0050] In addition, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the G2-4 or G2-4-27R polypeptide, as long as the altered polypeptide substantially retains the lipase activity of the original polypeptide.

[0051] As used herein, the term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids used in protein biosynthesis, as well as other amino acids, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethylthionine, etc., which are known to those skilled in the art. Amino acid analogs include modified forms of natural and non-naturally occurring amino acids. Such modifications may include, for example, replacing chemical groups and moieties on the amino acids, or derivatizing the amino acids. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge space characteristics of the amino acids. For example, an organic structure that mimics arginine (Arg or R) has a positively charged portion that is located in a similar molecular space and has the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures to maintain optimal spatial and charge interactions of the amino acids or amino acid functional groups. One skilled in the art can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0052] In some embodiments, variants of the amino acid sequence set forth in SEQ ID NO: 4 have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater identity with SEQ ID NO: 4. In a preferred embodiment, the polypeptide variant has greater than 99% identity with the sequence set forth in SEQ ID NO: 1.

[0053] In some embodiments, the variant of the amino acid sequence set forth in SEQ ID NO: 6 has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater homology to SEQ ID NO: 6. In a preferred embodiment, the polypeptide variant has greater than 99% homology to the sequence set forth in SEQ ID NO: 2.

[0054] As used herein, "homology" is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps, if necessary, to achieve the maximum percentage of homology. Methods and computer programs for alignment are well known in the art.

[0055] As used herein, "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and variants thereof and synthetic and naturally occurring analogs thereof. Thus, these terms apply to naturally occurring amino acid polymers and naturally occurring chemical derivatives thereof, as well as amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids (such as chemical analogs of corresponding naturally occurring amino acids). Such derivatives include, for example, post-translational modifications and degradation products, including phosphorylated, glycosylated, oxidized, isomerized, and deaminated variants of the polypeptide fragment set forth in SEQ ID NO: 4 or 6.

[0056] It is an established rule in protein chemistry that certain amino acid substitutions, known as "conservative amino acid substitutions," can occur frequently in proteins without altering the conformation or function of the protein.

[0057] Conservative amino acid substitutions in this application include, but are not limited to, substitutions of any one of glycine (G), alanine (A), isoleucine (I), valine (V) and leucine (L) for any other of these aliphatic amino acids; substitutions of threonine (T) with serine (S) and vice versa; substitutions of glutamate (E) with aspartic acid (D) and vice versa; substitutions of asparagine (N) with glutamine (Q) and vice versa; substitutions of arginine (R) with lysine (K) and vice versa; substitutions of any other of these aromatic amino acids with phenylalanine (F), tyrosine (Y) and tryptophan (W); and substitutions of cysteine ​​(C) with methionine (M) and vice versa. Other substitutions may also be considered conservative, depending on the specific amino acid context and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and valine (V). The relatively hydrophobic methionine (M) is frequently interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchanged at positions where the important characteristic of the amino acid residue is its charge and the difference in pK between the two residues is not significant. Still other changes can be considered "conservative" under certain circumstances (see, for example, BIOCHEMISTRY at pp. 13-15, 2 nd ed. Lubert Stryered. (Stanford University); Henikoff et al., Proc. Nat'l Acad. Sci. USA (1992) 89: 10915-10919; Lei et al., J. Biol. Chem. (1995) 270 (20): 11882-11886).

[0058] In certain embodiments, the polypeptide of the present application, such as the polypeptide shown in SEQ ID NO: 4 or 6 or a variant thereof, is fused with a heterologous polypeptide. In some embodiments, the fusion protein substantially retains the lipase activity of the polypeptide shown in SEQ ID NO: 4 or 6. In certain embodiments, the heterologous polypeptide is linked to the N-terminus of the polypeptide shown in SEQ ID NO: 4 or 6. In certain embodiments, the heterologous polypeptide is linked to the C-terminus of the polypeptide shown in SEQ ID NO: 4 or 6. In these embodiments, the heterologous polypeptide can be selected from a purification tag (for example, including but not limited to GST, MBP), an epitope tag (for example, including but not limited to Myc, FLAG), a targeting sequence, a signal peptide, and the like. In specific embodiments, the fusion protein comprises the polypeptide shown in SEQ ID NO: 4 or 6 and a tag, which is bound to the C-terminus or N-terminus of the polypeptide shown in SEQ ID NO: 4 or 6, typically a peptide tag.

[0059] The polypeptides of the present application maintain high enzymatic activity under acidic pH conditions, facilitating fatty acid methyl esterification. The "acidic pH" herein refers to a pH value less than 7, preferably less than 6.5, and more preferably less than 6. In some embodiments, the acidic pH can be 6, 5, 4, 3, 2, 1, or any value in between.

[0060] polynucleotides

[0061] The present application provides a polynucleotide encoding a polypeptide disclosed herein, which comprises or consists of a sequence selected from the following:

[0062] (a) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 or 6, or a sequence comprising at least one amino acid substitution, deletion or addition in SEQ ID NO: 4 or 6; and

[0063] (b) A nucleotide sequence that hybridizes to the nucleotide sequence in a) under stringent conditions.

[0064] In certain specific embodiments, the polynucleotides of the present application encode the polypeptides shown in SEQ ID NO: 4 or 6 and functionally equivalent variants thereof. In one embodiment, the polynucleotides of the present application have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with polynucleotides encoding the polypeptides shown in SEQ ID NO: 4 or 6 and functionally equivalent variants thereof.

[0065] In certain embodiments, the polynucleotide of the present application comprises a nucleotide sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the nucleotide sequence shown in SEQ ID NO: 3 or 5. In some embodiments, the polynucleotide of the present application comprises the nucleotide sequence shown in SEQ ID NO: 3 or 5.

[0066] In some embodiments, the polynucleotide of the present application consists of a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the nucleotide sequence shown in SEQ ID NO: 3 or 5. In a preferred embodiment, the polynucleotide of the present application consists of the nucleotide sequence shown in SEQ ID NO: 3 or 5. In a more preferred embodiment, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO: 5.

[0067] In a specific embodiment, the polynucleotide shown in SEQ ID NO:3 encodes the amino acid sequence shown in SEQ ID NO:4; the polynucleotide shown in SEQ ID NO:5 encodes the amino acid sequence shown in SEQ ID NO:6.

[0068] As used herein, the term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, cDNA, or DNA, including single-stranded and double-stranded forms of DNA. The term generally refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide.

[0069] In certain embodiments, the polynucleotide of the present application comprises a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence encoding a polypeptide as shown in SEQ ID NO: 4 or 6 and functionally equivalent variants thereof, or consists of a nucleotide sequence that specifically hybridizes with a nucleotide sequence encoding a polypeptide as shown in SEQ ID NO: 4 or 6 and functionally equivalent variants thereof and encodes a polypeptide that is functionally equivalent to the polypeptide as shown in SEQ ID NO: 4 or 6.

[0070] Those skilled in the art can routinely select stringent conditions for DNA hybridization. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strands are in an environment below their melting temperature. The higher the degree of homology between the probe and the hybridizable sequence, the higher the relative humidity that can be used. Thus, higher relative humidity often results in more stringent reaction conditions, while at lower temperatures, the stringency is lower. For a detailed description of stringent conditions for hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0071] In certain embodiments, stringent conditions for DNA hybridization include: 1) low ionic strength and high temperature during washing, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; 2) use of a denaturing agent such as formamide during hybridization, such as 50% (v / v) formamide plus 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polydiene pyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, and 750 mM sodium chloride and 75 mM sodium citrate at 42°C; or (3) overnight hybridization at 42°C in a hybridization solution containing 50% formamide, 5×SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.5), and 1% 5 ... 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 mg / mL), 0.1% SDS, and 10% dextran sulfate, followed by a wash in 0.2× SSC (sodium chloride / sodium citrate) at 42°C for 10 minutes, and a high stringency wash in 0.1× SSC containing EDTA at 55°C. Moderately stringent conditions can be determined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989. Moderately stringent conditions involve using wash solutions and hybridization conditions (such as temperature, ionic strength, and SDS percentage) that are less stringent than those described above. For example, moderately stringent conditions include hybridization with at least about 16% v / v to at least about 30% v / v formamide and at least about 0.5 M to at least about 0.9 M salt at 42°C, and a wash with at least about 0.1 M to at least about 0.2 M salt at 55°C. Moderately stringent conditions can also include hybridization with 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS at 65°C, and washing with (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 47.2), and 5% SDS at 60-65°C. Practitioners will adjust temperature, ionic strength, and other factors based on factors such as probe length. The degree of stringency when hybridizing nucleic acids depends on the length of the nucleic acid molecules and the degree of complementarity, as well as other variables well known in the art. The greater the similarity or homology between two nucleotide sequences, the greater the Tm of the nucleic acid hybrid containing those sequences. The relative stability of nucleic acid hybridizations (corresponding to higher Tm) decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. Preferably, the minimum length of hybridizable nucleic acids is at least about 12 nucleotides, preferably at least about 16, more preferably at least about 24, and most preferably at least about 36 nucleotides.

[0072] In some embodiments, the inventors of the present application used the lipase (TL) gene from Thermomyces lanuginosus as a basis, optimized the codons of the sequence encoding the mature protein according to the codon preference of Pichia pastoris, and expressed the synthesized gene using Escherichia coli or Pichia pastoris as the expression host.

[0073] The polynucleotides disclosed herein can be combined with other DNA sequences, such as promoters, polyadenylation signals, other restriction enzyme cleavage sites, multiple cloning sites, other coding segments, etc., so that their total length can vary significantly. It is therefore contemplated that polynucleotide fragments of almost any length can be utilized; the total length is preferably limited by the convenience of preparation and use in the intended recombinant DNA protocol.

[0074] Can utilize any one of the multiple mature techniques known in the art and obtainable to prepare, manipulate and / or express polynucleotide and its fusion.For example, the polynucleotide sequence encoding the polypeptide of the present application or its variant can be used in recombinant DNA molecule to instruct the expression of polypeptide in appropriate host cell.Due to the inherent degeneracy of genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences can also be used in the present application, and these sequences can be used to clone and express given polypeptide.

[0075] In addition, the polynucleotide sequences of the present application can be modified using methods well known in the art, including but not limited to changes in the cloning, processing, expression and / or activity of gene products.

[0076] In certain embodiments, the polynucleotides of the present application are produced by artificial synthesis, such as direct chemical synthesis or enzymatic synthesis. In alternative embodiments, the polynucleotides are produced by recombinant technology.

[0077] In a specific embodiment, the wild-type TL amino acid sequence used in this application is derived from NCBI (GenBank Accession: O59952). The DNA sequence SEQ ID NO: 1 was designed based on the codon preference of Pichia pastoris, and the DNA sequence was cloned into the pAO815 plasmid to obtain the pAO-TL expression vector.

[0078] In certain embodiments, the sequence of the obtained polynucleotide can be determined by conventional methods, preferably by the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). This type of polynucleotide sequence determination can also be completed using a commercial sequencing kit.

[0079] expression vector

[0080] The present application provides an expression vector comprising the polynucleotide of the present application.

[0081] The "expression vector" described herein is a recombinant or synthetic nucleic acid construct having a series of specific nucleic acid elements that allow transcription of a particular nucleic acid in a host cell. The expression vector used herein can be a plasmid vector such as pPIC9K, pAO815, pUC57, pET-24a(+), pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315, or a viral vector such as an adenovirus, an adeno-associated virus, a retrovirus, a semliki forest virus (sFv) vector, or other vectors well known in the art.

[0082] In certain embodiments, the polynucleotide sequence encoding the polypeptide shown in SEQ ID NO: 4 or 6 and variants thereof is cloned into a vector to construct a recombinant vector containing the polynucleotide described in the present application.

[0083] In a preferred embodiment, the expression vector used to clone the polynucleotide is a plasmid vector. In a more preferred embodiment, the plasmid vector is pPIC9K or pAO815.

[0084] In a specific embodiment, the above expression vector further comprises a regulatory sequence for regulating the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence.

[0085] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence required to achieve expression of the coding sequence to which it is linked. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and terminators; in eukaryotes, such regulatory sequences generally include promoters, terminators, and, in some cases, enhancers. Thus, the term "regulatory sequence" includes all sequences whose presence is minimally necessary for expression of a gene of interest, and may also include other sequences whose presence is advantageous for expression of a gene of interest, such as leader sequences.

[0086] As used herein, the term "operably linked" refers to a situation in which the sequences involved are in a relationship permitting them to function in their intended manner. Thus, for example, a regulatory sequence "operably linked" to a coding sequence allows expression of the coding sequence to be achieved under conditions compatible with the regulatory sequences.

[0087] In certain embodiments, expression vectors comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 4 or 6 and variants thereof and appropriate transcriptional / translational regulatory elements are constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like (Sambroook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). The nucleotide sequence is operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include the lac or trp promoters of Escherichia coli; the PL promoter of λ phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, retroviral LTRs, and other known promoters that control gene expression in prokaryotes or eukaryotes or their viruses. The expression vector may also include a ribosome binding site for translation initiation and a transcription terminator. Inserting an enhancer sequence into the vector will enhance transcription in higher eukaryotic cells. Enhancers are cis-acting factors in DNA expression, typically about 10 to 300 base pairs in length, that act on promoters to increase gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs on the late side of the replication origin), the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0088] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0089] host cells

[0090] The present application provides host cells comprising the polynucleotides or expression vectors disclosed herein.

[0091] In certain embodiments, a polynucleotide encoding the polypeptide shown in SEQ ID NO: 4 or 6 and variants thereof, or an expression vector containing the polynucleotide, is transformed or transduced into a host cell to obtain a genetically engineered host cell containing the polynucleotide or expression vector.

[0092] The host cells used herein can be any host cell well known to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Exemplary bacterial cells include any species of Escherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas, and Staphylococcus, including, for example, Escherichia coli, Lactococcus, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, and Pseudomonas fluorescens. Exemplary fungal cells include any species of Aspergillus. Exemplary yeast cells include any species of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, or Saccharomyces, including Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces. Exemplary insect cells include any species of Spodoptera litura or Drosophila, including Drosophila S2 and Spodoptera litura Sf9. Exemplary animal cells include CHO, COS, or melanoma, or any mouse or human cell line. Selecting an appropriate host is within the capabilities of those skilled in the art.

[0093] In a specific embodiment, the host cell used in this application is Escherichia coli. In a preferred embodiment, the expression vector carrying the polynucleotide sequence of this application is transformed into the Escherichia coli DH5α strain for induced expression. In another specific embodiment, the expression vector carrying the polynucleotide sequence of this application is transformed into Pichia pastoris cells for expression. Pichia pastoris strains that can be used in this application include GS115, M314, etc., which are suitable for general yeast transformation methods.

[0094] The expression vector can be introduced into the host cell using any technique known in the art, including transformation, transduction, transfection, viral infection, gene gun or Ti-mediated gene transfer. Specific methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). As an example, when the host is a prokaryotic organism such as Escherichia coli, competent cells can be harvested after the exponential growth phase and transformed using electroporation methods well known in the art.

[0095] In a specific embodiment, a recombinant expression vector containing a polynucleotide encoding a TL mutant is linearized with the SalI restriction endonuclease and then transformed into Pichia pastoris competent cells by electroporation. The transformants are then plated and cultured on MGYS plates. Transformants are then selected and cultured on BMMY-vinyl laurate selection plates. Finally, the transformant with the largest hydrolysis zone is selected for testing.

[0096] Methods for screening and preparing polypeptides of the present application

[0097] The polypeptides of the present application can be screened and prepared by any suitable method known to those skilled in the art.

[0098] In some embodiments, the polypeptides disclosed herein are obtained by performing site-directed mutagenesis on TL, and the results can be verified and used to guide the directed modification of TL.

[0099] In some embodiments, the polypeptide of the present application can also be produced by recombinant technology, or chemical synthesis. The method of producing recombinant peptides is known in the art. The chemical synthesis method of peptides is also well known to those skilled in the art. For example, the polypeptide of the present application and variants thereof (Merrifield, J.Am.Chem.Soc.85:2149-2154 (1963)) can be produced by the directed peptide synthesis using solid phase technology. Protein synthesis can be performed manually or by automation. For example, automated synthesis can be achieved using the 431A peptide synthesizer (Perkin Elmer) of Applied Biosystems. Alternatively, different sizes can be chemically synthesized and combined to prepare desired molecules using chemical methods.

[0100] In some embodiments, the screening method for the polypeptides disclosed herein comprises:

[0101] 1) mutating the nucleotide sequence shown in SEQ ID NO: 1 to obtain a mutant sequence,

[0102] 2) The mutant sequence obtained in step 1) is cloned into an expression vector and then transformed or transduced into a suitable host cell,

[0103] 3) culturing the host cells in step 2), recovering the recombinant expression vector, and then transforming the product into a yeast strain for cultivation, and

[0104] 4) Screening for mutant polypeptides having higher enzyme activity and / or acid resistance than the polypeptide shown in SEQ ID NO: 2.

[0105] In a specific embodiment, the DNA sequence set forth in SEQ ID NO:1 is cloned into the pAO815 plasmid to generate the pAO-TL expression vector. Error-prone PCR is performed using the pAO-TL vector as a template, using TaKaRa Taq enzyme and primer pairs, to generate a collection of mutant amplicon fragments. The resulting fragments are cloned into pAO-TL via the HindIII and EcoRI restriction sites, and the resulting vector is transformed into the Escherichia coli DH5α strain. The E. coli clones are washed and cultured in LB liquid medium. The plasmids are extracted, linearized with SalI, and then transformed into Pichia pastoris competent cells by electroporation. The transformants are plated and cultured on MGYS plates to generate a TL library of Pichia pastoris mutants. Single colonies from the plates are selected and plated on BMMY-vinyl laurate screening plates. Colonies with large color change zones are selected. In an alternative embodiment, the screened clones undergo a second round of random mutagenesis, using similar procedures as above, to obtain further mutant lipases. By measuring the enzyme activity under different conditions, mutants with high enzyme activity and / or high acid resistance are screened.

[0106] In some embodiments, the verified mutant is sequenced to determine the sequence after mutation.

[0107] A suitable host cell refers to a host cell suitable for the expression of an expression vector or a target polynucleotide. A suitable culture medium refers to a culture medium suitable for the growth of host cells or for inducing expression thereof.

[0108] In certain embodiments, according to used host cell, various conventional culture media can be selected.Under the condition that is suitable for host cell growth, cultivate.Preferably, the host cell of through engineering approaches can be cultivated in the conventional nutrient medium that is modified to be suitable for activating promoter, to screen transformant or amplify the polynucleotide of the application.Convert suitable host cell and after host cell grows to suitable cell density, induce the promoter of selection with suitable method (such as temperature conversion or chemical induction), with cell recultivation for a period of time, to allow it to produce purpose polypeptide or its fragment.

[0109] In certain embodiments, the polypeptide produced by the host cell can be encapsulated in the cell, expressed on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. For example, the expressed polypeptide or its fragment can be recovered and purified from the recombinant cell culture by the following methods well known in the art: conventional renaturation treatment, protein precipitation agent treatment (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods. As an exemplary illustration, affinity chromatography purification of proteins containing peptide tags (such as His-tags, etc.) at the C-terminus or N-terminus is a conventional method for obtaining high-purity polypeptide preparations.

[0110] Application of polypeptides with lipase activity

[0111] The polypeptide of the present application is a polypeptide having lipase activity and can produce biodiesel by fatty acid methyl esterification. In some embodiments, the polypeptide of the present application is contacted with a fat feedstock and a lower alcohol to prepare biodiesel.

[0112] In some embodiments, the above-mentioned grease raw material is a high acid value grease raw material, i.e., a grease raw material with a high fatty acid content. For example, the grease raw material utilized can be waste cooking oil, acidified oil, soybean oil deodorized distillate, palm oil deodorized distillate (PFAD), animal fat, waste cooking oil or yellow grease, etc. In a preferred embodiment, the grease raw material is selected from waste cooking oil, acidified oil, palm oil fatty acid distillate (PFAD) or a mixture thereof.

[0113] In some embodiments, the lower alcohol is selected from methanol, ethanol, propanol, butanol and pentanol or any combination thereof. In a preferred embodiment, the lower alcohol is methanol.

[0114] The polypeptides disclosed herein have high enzymatic activity and / or acid resistance. In some embodiments, the enzymatic activity of the polypeptides disclosed herein (e.g., the polypeptides set forth in SEQ ID NO: 4 or 6) is higher than that of wild-type TL (e.g., the control polypeptide set forth in SEQ ID NO: 2) under acidic pH conditions (e.g., pH less than 5). In some embodiments, the polypeptides disclosed herein (e.g., the polypeptides set forth in SEQ ID NO: 4 or 6) can significantly reduce the free fatty acid content in the reaction system compared to wild-type TL (e.g., the control polypeptide set forth in SEQ ID NO: 2), making them suitable for further production of biodiesel.

[0115] Throughout the specification and claims, the words “comprises,” “comprising,” and “including” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0116] It will be appreciated that features, characteristics, components, or steps described in conjunction with a particular aspect, embodiment, or example of the present application may be applicable to any other aspect, embodiment, or example described herein unless incompatible therewith.

[0117] The above disclosure generally describes the present application, and the present application is further illustrated by the following examples. These examples are described only to illustrate the present application, rather than to limit the scope of the present application. Although special terms and values ​​are used herein, these terms and values ​​are also understood to be exemplary and do not limit the scope of the present application.

[0118] Example

[0119] Experimental Materials

[0120] 1) Experimental strains and plasmids

[0121] Strains: Pichia pastoris GS115 (Invitrogen), Escherichia coli DH5α (TAKARA).

[0122] Plasmid: pAO815 plasmid, purchased from Invitrogen, catalog number V18020.

[0123] 2) Culture media and solutions

[0124] LB liquid medium: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0.

[0125] LB solid medium: Add agar to LB liquid medium at a concentration of 1.5%.

[0126] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose.

[0127] YPD solid medium: Add agar to LB liquid medium at a concentration of 2%.

[0128] BMMY-vinyl laurate screening medium: Component A: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB) containing ammonium sulfate but not amino acids, 1% methanol (added after sterilization), 4×10 -5% D-biotin (added after sterilization), 0.1 M citric acid-sodium citrate buffer, pH 6.6 or pH 5.0, and 2% agar. Component B: Vinyl laurate substrate solution: Measure 150 ml of 4% PVA solution, add 50 ml of olive oil, and emulsify using a high-speed homogenizer at 8000 rpm for 3 minutes, pause for 1 minute, and then emulsify for another 3 minutes to prepare the substrate solution. Mix 100 ml of sterilized Component A with 12 ml of Component B, and add 1 ml of 0.1% rhodamine B.

[0129] BMGY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB) containing ammonium sulfate but not amino acids, 1% glycerol, 4×10-5% D-biotin, 0.1 M citric acid-sodium citrate buffer, pH 6.6.

[0130] Improved Bradford protein concentration determination kit (purchased from Shanghai Shenggong Bioengineering Co., Ltd.)

[0131] Restriction endonuclease EcoRI (purchased from New England Biotechnology (Beijing) Co., Ltd.)

[0132] PCR enzyme: TaKaRa Taq, HS DNA Polymerase (purchased from Takara Biotechnology (Dalian) Co., Ltd.)

[0133] T4 DNA ligase (purchased from Fuzyme Technologies Co., Ltd.)

[0134] Example 1: Construction of TL mutation library and mutant screening

[0135] The amino acid sequence of TL was based on the wild-type Thermomyces lanuginosus lipase amino acid sequence, which was obtained from NCBI (GenBank Accession: O59952). The DNA sequence SEQ ID NO: 1 was designed based on the codon preference of Pichia pastoris, and the α-mating factor leader peptide sequence was added. This sequence was derived from the DNA sequence of the commercial vector pPIC9K. The DNA sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd. and cloned into the pAO815 plasmid to generate the pAO-TL expression vector.

[0136] Using the pAO-TL vector as a template, TaKaRa Taq enzyme and primers TL-1 (5'-GCGCCTAGGCCGCGGCGAAACGATGAGATTTCCTTCAATTTTTAC-3'; SEQ ID NO: 7) / TL-2 (5'-CCGGAATTCTTACAAGCAAGTACCAATCAG-3'; SEQ ID NO: 8) were used for error-prone PCR (0.3 mM MnCl2 was additionally added during PCR) to obtain a collection of mutant amplicon fragments of approximately 755 bp in size. The resulting fragments were cloned into pAO-TL via the HindIII and EcoRI restriction sites, and the resulting vector was transformed into the Escherichia coli DH5α strain, resulting in a total of 1×10 6 TL mutants.

[0137] Transform the plasmid library into Escherichia coli DH5α strain. Wash all E. coli clones into LB liquid medium (containing 100 μg / ml ampicillin) and culture at 37°C for 4 hours. Extract the plasmid, linearize with SalI, and recover an approximately 8.5 kb fragment. Take 500 ng of the vector and transform it into competent cells of Pichia pastoris GS115 strain using electroporation. Inoculate the transformants onto MGYS plates and culture at 30°C for 3 days to obtain the TL Pichia pastoris mutant library. Pick single colonies from the plate and transfer them to BMMY-vinyl laurate screening plates. Select the clone with the largest color change circle. Clone number G2-4.

[0138] The G2-4 strain was inoculated into 3 ml YPD liquid medium and cultured at 30°C overnight to extract genomic DNA. HS DNA polymerase and primer pair AOX1-5 (5'-GACTGGTTCCAATTGACAACG-3'; SEQ ID NO: 9) / AOX1-3 (5'-GGCAAATGGCATTCTGACATCCTC-3'; SEQ ID NO: 10) were used for PCR amplification to obtain the DNA sequence of the lipase in the G2-4 strain. The obtained sequence was sent to Shanghai Shenggong Biotechnology Co., Ltd. and sequenced using primer pair AOX1-5 / AOX1-3. The DNA sequencing results of the lipase of the G2-4 strain are shown in SEQ ID NO: 3. In the amino acid sequence of the lipase of the G2-4 strain (SEQ ID NO: 4), asparagine at position 33 was mutated to lysine, glycine at position 38 was mutated to cysteine, aspartic acid at position 96 was mutated to arginine, aspartic acid at position 111 was mutated to alanine, serine at position 231 was mutated to glutamic acid, and asparagine at position 233 was mutated to arginine.

[0139] Example 2: Second round of random mutagenesis of G2-4

[0140] The G2-4 strain was inoculated into 3 ml YPD liquid medium and cultured at 30°C overnight to extract genomic DNA. HS DNA polymerase and primers were used to amplify TL-1 / TL-2 by PCR to obtain the DNA sequence of lipase in the G2-4 strain, which was digested with SacII and EcoRI. After recovery, it was cloned into the pAO-TL vector digested with SacII and EcoRI to obtain the pAO-G2-4 vector.

[0141] Using the pAO-G2-4 vector as a template, TaKaRa Taq enzyme and primers were used to perform error-prone PCR on TL-1 / TL-2 (0.3 mM MnCl2 was added during PCR), resulting in a collection of mutant amplicon fragments of approximately 755 bp in size. The resulting fragments were cloned into pAO-TL through the HindIII and EcoRI restriction sites, and the resulting vector was transformed into the Escherichia coli DH5α strain, resulting in a total of 1×10 6 G2-4 mutants.

[0142] Transform the plasmid library into Escherichia coli DH5α strain, wash all E. coli clones into LB liquid medium (containing 100μg / ml ampicillin), and culture at 37℃ for 4h. Extract the plasmid, linearize with SalI, and recover a fragment of about 8.5kb. Take 500ng of the vector and transform it into the competent cells of Pichia pastoris M314 strain by electroporation. Inoculate the transformant on the MGYS plate and culture at 30℃ for 3 days to obtain the TL Pichia pastoris mutant library. Pick a single clone on the plate and transfer it to the BMMY-vinyl laurate screening plate at pH 5.0. Select the clone with the largest color change circle. Inoculate the clone into 3ml YPD liquid medium, culture overnight at 30℃, and extract the genomic DNA. Use this genome as a template HS DNA polymerase and primers were used to perform PCR amplification on AOX1-5 / AOX1-3 to obtain the DNA sequence of the lipase from the mutant strain. The resulting sequence was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing using primers for AOX1-5 / AOX1-3. The DNA sequencing results of the lipase from the mutant strain are shown in SEQ ID NO: 5. The amino acid sequence of the mutant (SEQ ID NO: 6) showed that aspartic acid at position 27 was mutated to arginine, asparagine at position 33 was mutated to lysine, glycine at position 38 was mutated to cysteine, aspartic acid at position 111 was mutated to alanine, serine at position 231 was mutated to glutamic acid, and asparagine at position 233 was mutated to arginine. Compared to the sequence of G2-4, this mutant had a mutation at amino acid position 27, from aspartic acid to arginine, and was therefore named the G2-4-27R polypeptide.

[0143] Example 3: Mutant performance testing

[0144] The G2-4-27R and G2-4 mutants and the wild-type TL expression strain were activated in liquid YPD and then inoculated into BMGY medium. The culture was cultured overnight at 30°C with shaking at 220 rpm. The culture was transferred to BMMY medium with an initial OD600 of 6.

[0145] Initially, the cells were induced with 2% methanol. 1% methanol was added after 24 and 32 hours, and after 48 and 56 hours, respectively. Samples were collected after 72 hours. The resulting samples were desalted and concentrated 40-fold using ultrafiltration tubing with a 10 kDa molecular weight cutoff. The treated samples were then added to a buffer solution (20 mM citric acid-sodium citrate buffer, pH 6.6).

[0146] Prepare 1 mg / ml of TL, G2-4, and G2-4-27R enzyme solutions. Place one aliquot in a 45°C water bath. Dilute the other aliquot with pH 4.2 citric acid-sodium citrate buffer and store at 45°C. Samples were taken at 4 and 22 hours and assayed for activity by acid-base titration using olive oil as a substrate. The activity of the enzyme solution at room temperature was used as a control and recorded as 100%. The enzyme activity assay was performed as follows:

[0147] Measure 150 ml of 4% PVA solution, add 50 ml of olive oil, and emulsify using a high-speed homogenizer at 8000 rpm for 3 minutes. Pause for 1 minute and then emulsify for another 3 minutes to prepare the substrate solution (this solution must be prepared and used immediately).

[0148]

[0149] The results are as follows Figure 1As shown in Figure 2, the enzyme activities of TL, G2-4, and G2-4-27R were very stable after being stored in citric acid-sodium citrate buffer at pH 6.6 at 45°C for 22 hours, with no significant changes. However, at the same pH of 4.2 at 45°C, after 22 hours, the residual enzyme activity of wild-type TL was only 8%, while that of G2-4 was 42% and that of G2-4-27R was 60%. Figure 2 shown.

[0150] Example 4: Effect of methyl esterification reaction of mutants

[0151] The G2-4-27R and G2-4 mutants and the wild-type TL expression strain were activated in liquid YPD and then inoculated into BMGY medium. The culture was cultured overnight at 30°C with shaking at 220 rpm. The culture was transferred to BMMY medium with an initial OD600 of 6.

[0152] Initially, the cells were induced with 2% methanol. 1% methanol was added after 24 and 32 hours, and after 48 and 56 hours, respectively. Samples were collected after 72 hours. The resulting samples were desalted and concentrated 40-fold using ultrafiltration tubing with a 10 kDa molecular weight cutoff. The treated samples were then added to a buffer solution (20 mM citric acid-sodium citrate buffer, pH 6.6).

[0153] Prepare 30 mg / ml of enzyme solution of TL, G2-4 and G2-4-27R. Use the dosage of each component as shown in the table below to prepare biodiesel.

[0154]

[0155] The results are as follows Figure 3 As shown, both G2-4 and G2-4-27R significantly reduced the free fatty acid content in the reaction system compared to wild-type TL. For example, after 24 hours of reaction, G2-4-27R reduced the free fatty acid content in the reaction system to approximately 3.4%, meeting the standard for further biodiesel production. However, when treated with the wild-type TL at the same enzyme dosage, the free fatty acid content in the reaction system remained as high as 60% after 24 hours, making further biodiesel production impossible.

[0156] It will be understood that although the present application has been described in certain forms, the present application is not limited to what is shown and described in this specification. It will be apparent to those skilled in the art that various modifications may be made without departing from the scope of the present application. Such modifications are within the scope of the present application. Sequence Listing <110> Wilmar (Shanghai) Biotechnology R&D Center Co., Ltd. <120> Acid-resistant lipase <130> 19C14017CN <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 810 <212> DNA <213> Thermomyces lanuginosus <400> 1 gaagtctctc aagacttgtt caaccagttc aacttgttcg ctcaatactc tgccgctgcc 60 tactgtggta agaacaatga tgctccagct ggtactaaca ttacctgtac tggtaacgct 120 tgtccagaag ttgagaaggc tgatgctacc ttcctgtact ccttcgaaga ctctggagtt 180 ggagatgtta ctggtttcct ggccttggat aacactaaca agttgatcgt tctgtccttc 240 agaggttcca gatccatcga gaactggatt ggtaacttga actttgactt gaaggagatc 300 aacgacatct gttctggatg tcgtggtcac gatggattta cctcctcttg gagatctgtt 360 gctgatacct tgagacagaa ggtcgaagat gctgtcagag aacatccaga ctatagagtt 420 gtcttcactg gtcactcctt gggaggtgcc ttggctactg ttgctggtgc tgacttgcgt 480 ggtaatggtt atgacattga tgtcttctcc tacggtgctc caagagttgg taatcgtgcc 540 ttcgctgagt ttctgaccgt ccaaactgga ggtactttgt acagaattac ccatactaac 600 gacattgttc caagattgcc accacgtgag ttcggatact ctcattcctc tccagagtac 660 tggatcaagt ctggaacctt ggttccagtc actcgtaacg acatcgtcaa gattgaaggt 720 attgatgcca ctggaggtaa caatcaacca aacattccag acattccagc tcacttgtgg 780 tactttggtc tgattggtac ttgcttgtaa 810 <210> 2 <211> 269 <212> PRT <213> (Thermomyces lanuginosus) <400> 2 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Asp 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 3 <211> 810 <212> DNA <213> Artificial Sequence <400> 3 gaagtctctc aagacttgtt caaccagttc aacttgttcg ctcaatactc tgccgctgcc 60 tactgtggta agaacaatga tgctccagct ggtactaaga ttacctgtac ttctaacgct 120 tgtccagaag ttgagaaggc tgatgctacc ttcctgtact ccttcgaaga ctctggagtt 180 ggagatgtta ctggtttcct ggccttggat aacactaaca agttgatcgt tctgtccttc 240 agaggttcca gatccatcga gaactggatt ggtaacttga actttaggtt gaaggagatc 300 aacgacatct gttctggatg tcgtggtcac gcgggattta cctcctcttg gagatctgtt 360 gctgatacct tgagacagaa ggtcgaagat gctgtcagag aacatccaga ctatagagtt 420 gtcttcactg gtcactcctt gggaggtgcc ttggctactg ttgctggtgc tgacttgcgt 480 ggtaatggtt atgacattga tgtcttctcc tacggtgctc caagagttgg taatcgtgcc 540 ttcgctgagt ttctgaccgt ccaaactgga ggtactttgt acagaattac ccatactaac 600 gacattgttc caagattgcc accacgtgag ttcggatact ctcattcctc tccagagtac 660 tggatcaagt ctggaacctt ggttccagtc gagcgtcgtg acatcgtcaa gattgaaggt 720 attgatgcca ctggaggtaa caatcaacca aacattccag acattccagc tcacttgtgg 780 tactttggtc tgattggtac ttgcttgtaa 810 <210> 4 <211> 269 <212> PRT <213> Artificial Sequence <400> 4 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30[[ID=2⑨]] Lys Ile Thr Cys Thr Cys Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Arg 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Ala Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Glu Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 5 <211> 810 <212> DNA <213> Artificial Sequence <400> 5 gaagtctctc aagacttgtt caaccagttc aacttgttcg ctcaatactc tgccgctgcc 60 tactgtggta agaacaatag agctccagct ggtactaaga ttacctgtac ttctaacgct 120 tgtccagaag ttgagaaggc tgatgctacc ttcctgtact ccttcgaaga ctctggagtt 180 ggagatgtta ctggtttcct ggccttggat aacactaaca agttgatcgt tctgtccttc 240 agaggttcca gatccatcga gaactggatt ggtaacttga actttaggtt gaaggagatc 300 aacgacatct gttctggatg tcgtggtcac gcgggattta cctcctcttg gagatctgtt 360 gctgatacct tgagacagaa ggtcgaagat gctgtcagag aacatccaga ctatagagtt 420 gtcttcactg gtcactcctt gggaggtgcc ttggctactg ttgctggtgc tgacttgcgt 480 ggtaatggtt atgacattga tgtcttctcc tacggtgctc caagagttgg taatcgtgcc 540 ttcgctgagt ttctgaccgt ccaaactgga ggtactttgt acagaattac ccatactaac 600 gacattgttc caagattgcc accacgtgag ttcggatact ctcattcctc tccagagtac 660 tggatcaagt ctggaacctt ggttccagtc gagcgtcgtg acatcgtcaa gattgaaggt 720 attgatgcca ctggaggtaa caatcaacca aacattccag acattccagc tcacttgtgg 780 tactttggtc tgattggtac ttgcttgtaa 810 <210> 6 <211> 269 <212> PRT <213> Artificial Sequence <400> 6 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Arg Ala Pro Ala Gly Thr 20 25 30 Lys Ile Thr Cys Thr Cys Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Arg 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Ala Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Glu Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <400> 7 gcgcctaggc cgcggcgaaa cgatgagatt tccttcaatt tttac 45 <2!10> 8 <211> 30 <212> DNA <213> Artificial Sequence <400> 8 ccggaattct tacaagcaag taccaatcag 30 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <400> 9 gactggttcc aattgacaac g 21 <210> 10 <211> 24 <212> DNA It should be noted that there seems to be an error in the original text where <2!10> appears. It is retained as is in the translation but should be corrected in the original if it is a typo.<213> Artificial Sequence <400> 10 ggcaaatggc attctgacat cctc 24

Claims

1. A polypeptide having lipase activity, which is selected from the amino acid sequences shown in SEQ ID NO: 4 or 6.

2. A polynucleotide encoding the polypeptide according to claim 1.

3. The polynucleotide according to claim 2, which is selected from the nucleotide sequences shown in SEQ ID NO: 3 or 5.

4. An expression vector comprising the polynucleotide according to claim 2 or 3.

5. The expression vector according to claim 4, which further comprises a regulatory sequence for regulating the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence.

6. The expression vector according to claim 5, wherein the expression vector is a plasmid.

7. A host cell comprising the polynucleotide according to claim 2 or 3, or the expression vector according to claim 5 or 6, and the host cell is not a plant cell.

8. The host cell according to claim 7, wherein the host cell is yeast or Escherichia coli.

9. Use of the polypeptide according to claim 1 for carrying out a fatty acid methyl esterification reaction or for preparing biodiesel.

10. A method for preparing biodiesel, which comprises contacting the polypeptide according to claim 1 with an oil raw material and a lower alcohol.

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