A new monoglyceride-diacylglycerol lipase

Abstract

The present invention provides a polypeptide having lipase activity. The polypeptide provided by the present invention comprises a sequence selected from the following or consists of a sequence selected from the following: (a) the amino acid sequence shown in SEQ ID NO: 2, and (b) a sequence obtained by substituting, deleting or adding at least one amino acid to the sequence described in (a), wherein the polypeptide variant obtained from (b) still retains lipase activity. The polypeptide provided by the present invention can hydrolyze and / or synthesize monoglycerides and / or diglycerides.

Description

Field of the Invention

[0001] This application belongs to the field of enzyme engineering. Specifically, it relates to polypeptides with lipase activity, nucleic acids encoding the same, expression vectors containing the encoding nucleic acids, and host cells. Background of the Invention

[0002] Lipase is a type of esterase that can catalyze the hydrolysis of ester bonds in triglycerides, diglycerides, monoglycerides, other small molecule esters, polyol esters, and polyacid esters. Fat is the natural substrate of lipase. During the hydrolysis process, diglycerides and monoglycerides are produced, and the final hydrolysis products are glycerol and fatty acids. Lipase has mild hydrolysis conditions, few by-products, and does not require coenzymes. Since fats are mostly hydrophobic substances, the hydrolysis reaction occurs at the oil-water interface or in an organic phase. Mono-diacylglycerol lipase (MDGL) is a type of lipase. MDGL has substrate specificity and only acts on monoglyceride (MAG) and diglyceride (DAG), and does not catalyze triglyceride (TAG). It can use esterification or transesterification reactions to produce monoglycerides with high industrial value.

[0003] Yamaguchi et al. first obtained MDGL from Penicillium camemberti. MDGL has two forms with molecular weights of 37KDa and 39KDa respectively. MDGL has a signal peptide composed of 26 amino acids, and the mature peptide contains 279 amino acids. Yamaguchi et al. measured the enzyme activity unit of MDGL from Penicillium camemberti using vinyl laurate as the substrate. After purification steps such as medium filtration, ethanol purification, ammonium sulfate precipitation, DEAE agarose gel filtration, cellulose membrane purification, and ion exchange, the specific enzyme activity of MDGL was 6680 U / mg. MDGL is encoded by mdlA, and mdlA has been expressed in Pichia pastoris.

[0004] MAG is an excellent emulsifier and has wide applications in the food, pharmaceutical, and cosmetic industries. The traditional synthesis process of MAG is to carry out continuous esterification of oil / glyceride with glycerol under high temperature, using inorganic base as a catalyst under nitrogen conditions. The product is a mixture of MAG, DAG, and TAG, and MAG is obtained by distillation, with a yield of MAG of 40 - 50%. Different from the traditional synthesis process, the enzymatic synthesis of MAG uses fatty acid and glycerol as substrates and catalyzes the synthesis of MAG under mild conditions. In enzymatic catalysis, the conversion rate of fatty acid exceeds 97%, and the proportion of MAG in the product exceeds 74%. The main problems restricting the application of enzymatic methods currently are the low activity of the enzyme and the high cost of the enzyme.

[0005] Therefore, there is an urgent need in the art to develop enzymes with higher activity. Summary of the Invention

[0006] The present invention provides a polypeptide with lipase activity.

[0007] The polypeptide of the present invention comprises a sequence selected from the following or consists of a sequence selected from the following:

[0008] (a) The amino acid sequence shown in SEQ ID NO:2, and

[0009] (b) A sequence obtained by substituting, deleting or adding at least one amino acid to the sequence described in (a),

[0010] wherein the polypeptide variant obtained from (b) still retains lipase activity.

[0011] In a specific embodiment of the present invention, the polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO:2. Preferably, the polypeptide consists of the amino acid sequence shown in SEQ ID NO:2.

[0012] The lipase activity of the present invention is monoglyceride-diglyceride lipase activity.

[0013] The present invention also provides a polynucleotide encoding the polypeptide of the present invention, which comprises a sequence selected from the following or consists of a sequence selected from the following:

[0014] (a) A nucleotide sequence encoding the amino acid sequence described in claim 1(a) or 1(b); and

[0015] (b) A nucleotide sequence that hybridizes with the nucleotide sequence in (a) under stringent conditions.

[0016] In a specific embodiment of the present invention, the polynucleotide provided by the present invention comprises the nucleotide sequence shown in SEQ ID NO:1. In a specific embodiment of the present invention, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO:1.

[0017] The present invention also provides an expression vector comprising at least one of the foregoing polynucleotides.

[0018] In a specific embodiment of the present invention, the expression vector of the present invention further comprises a regulatory sequence for regulating the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence.

[0019] The present invention also provides a host cell comprising the foregoing polynucleotide or expression vector. In a specific embodiment of the present invention, the host cell is yeast.

[0020] The present invention also provides the use of the foregoing polypeptide for the hydrolysis and / or synthesis of monoglyceride and / or diglyceride.

[0021] The present invention also provides a method for hydrolyzing and / or synthesizing monoglycerides and / or diglycerides. The method provided by the present invention is catalyzed by the aforementioned polypeptide, or by using the fermentation broth, supernatant or concentrate of the aforementioned host cell.

[0022] In a specific embodiment of the present invention, the substrate for the catalysis is p-nitrophenyl palmitate (pNPP). Brief Description of the Drawings

[0023] Figure 1 Shows the protein production results of the flask fermentation of the strain. Among them, CM1 is CTLmc production strain 1, CM2 is CTLmc production strain 2, and CM3 is CTLmc production strain 3.

[0024] Figure 2 Shows the pNPP hydrolysis activity results of the CTLmc flask fermentation protein. Among them, CM1 is the enzyme produced by CTLmc production strain 1, CM2 is the enzyme produced by CTLmc production strain 2, and CM3 is the enzyme produced by CTLmc production strain 3.

[0025] Figure 3 Shows the TLC analysis results of the hydrolysis products of CTLmc mono- and dioleate glycerides. Among them, 1 is the oleic acid standard; 2 is the 1-monoglyceride oleate standard; 3 is the 2-monoglyceride oleate standard; 4 is the blank sample; 5 is the 1,3-dioleate glyceride standard; 6 is the olein standard; 7 is the hydrolysis product of monoglyceride oleate CM1; 8 is the unhydrolyzed monoglyceride oleate; 9 is the hydrolysis product of dioleate glyceride CM1; 10 is the unhydrolyzed dioleate glyceride. Detailed Description of the Invention

[0026] The polypeptide of the present application

[0027] The present application provides a polypeptide with lipase activity, which comprises a sequence selected from the following or consists of a sequence selected from the following:

[0028] (a) The amino acid sequence shown in SEQ ID NO:2, and

[0029] (b) A sequence obtained by substituting, deleting or adding at least one amino acid to the sequence described in (a), wherein the polypeptide variant obtained from (b) still retains lipase activity.

[0030] The lipase activity of the present invention is monoglyceride-diacylglycerol lipase activity.

[0031] In certain embodiments, the number of the above-mentioned amino acid substitutions, deletions or additions is 1-30, preferably 1-20, more preferably 1-10, and the polypeptide variant obtained therein substantially retains the lipase activity of the unchanged protein.

[0032] In a preferred embodiment, the above polypeptide variant differs from the amino acid sequence shown in SEQ ID NO:2 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 above polypeptide variant differs from the amino acid sequence shown in SEQ ID NO:2 by about 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions.

[0033] In some embodiments, the above polypeptide consists of the amino acid sequence shown in SEQ ID NO:2. In a preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO:2.

[0034] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more conservative amino acid substitutions occur in the amino acid sequence of the polypeptide. The polypeptide after conservative substitution substantially retains the lipase activity of the original polypeptide.

[0035] In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can be deleted from the C-terminus or N-terminus of the polypeptide, and still have lipase activity.

[0036] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can also be added to the C-terminal or N-terminal region of the polypeptide, and the resulting polypeptide variant still has lipase catalytic activity.

[0037] In addition, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can be added or deleted from a region other than the C-terminus or N-terminus of the polypeptide, as long as the modified polypeptide substantially retains the lipase activity of the original polypeptide.

[0038] 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, ethylthreonine, etc., which are known to those skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substituting chemical groups and moieties on the amino acid, or derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge-space characteristics of an amino acid. For example, an organic structure mimicking arginine (Arg or R) has a positively charged moiety located in a similar molecular space and having 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 acid or amino acid functional group. Those skilled in the art can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0039] In some embodiments, variants of the amino acid sequence set forth in SEQ ID NO:2 have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO:2. In a preferred embodiment, the polypeptide variant has 99% or more homology to the sequence set forth in SEQ ID NO:1.

[0040] "Homology" as described herein is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence 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.

[0041] The "polypeptides" and "proteins" of this application are used interchangeably herein and refer to polymers of amino acid residues, as well as variants and synthetic and naturally occurring analogs thereof. Thus, these terms apply to naturally occurring amino acid polymers and their naturally occurring chemical derivatives, 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 the 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 polypeptide fragments set forth in SEQ ID NO:2.

[0042] It is an established principle in protein chemistry that certain amino acid substitutions, known as "conservative amino acid substitutions," can occur frequently in a protein without altering the conformation or function of that protein.

[0043] Conservative amino acid substitutions in this application include, but are not limited to, substituting any one of these aliphatic amino acids with any other of glycine (G), alanine (A), isoleucine (I), valine (V), and leucine (L); substituting serine (S) for threonine (T) and vice versa; substituting aspartic acid (D) for glutamic acid (E) and vice versa; substituting glutamine (Q) for asparagine (N) and vice versa; substituting lysine (K) for arginine (R) and vice versa; substituting any one of these aromatic amino acids with any other of phenylalanine (F), tyrosine (Y), and tryptophan (W); and substituting methionine (M) for cysteine (C) and vice versa. Other substitutions may also be considered conservative, depending on the particular amino acid environment and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchanged, as can alanine (A) and valine (V). The relatively hydrophobic methionine (M) can often be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchanged at positions where the important feature of the amino acid residue is its charge, and the different pKs of these two amino acid residues are not significant. In certain environments, still other changes can be considered "conservative" (see, for example, BIOCHEMISTRY at pp.13-15,2 nd ed. Lubert Stryer (Stanford University); Henikoff et al., Proc. Nat’l Acad. Sci. USA 0(1992) 89:10915-10919; Lei et al., J. Biol. Chem. (1995) 270(20):11882-11886).

[0044] In certain embodiments, the polypeptides of the present application, such as the polypeptide shown in SEQ ID NO:2 or its variants, are fused with a heterologous polypeptide. In some embodiments, the fusion protein substantially retains the lipase activity of the polypeptide shown in SEQ ID NO:2. In certain embodiments, the heterologous polypeptide is linked to the N-terminus of the polypeptide shown in SEQ ID NO:2. In certain embodiments, the heterologous polypeptide is linked to the C-terminus of the polypeptide shown in SEQ ID NO:2. In these embodiments, the heterologous polypeptide can be selected from purification tags (e.g., which can include but are not limited to: GST, MBP), epitope tags (e.g., which can include but are not limited to: Myc, FLAG), targeting sequences, signal peptides, etc. In a specific embodiment, the fusion protein comprises the polypeptide shown in SEQ ID NO:2 and a tag, which binds to the C-terminus or N-terminus of the polypeptide shown in SEQ ID NO:2, usually a peptide tag.

[0045] The polypeptides of the present application have the ability to hydrolyze and / or synthesize monoglycerides and / or diglycerides.

[0046] Polynucleotide

[0047] The present application provides polynucleotides encoding the polypeptides disclosed herein, which comprise a sequence selected from the following or consist of a sequence selected from the following:

[0048] (a) a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO:2, or a sequence that contains at least one amino acid substitution, deletion or addition in SEQ ID NO:2; and

[0049] (b) a nucleotide sequence that hybridizes with the nucleotide sequence in (a) under stringent conditions.

[0050] In certain specific embodiments, the polynucleotides of the present application encode the polypeptide shown in SEQ ID NO:2 and its functionally equivalent variants. 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 the polynucleotides encoding the polypeptide shown in SEQ ID NO:2 and its functionally equivalent variants.

[0051] In certain embodiments, the polynucleotides of the present application comprise a nucleotide sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the polynucleotides of the present application comprise the nucleotide sequence shown in SEQ ID NO:1.

[0052] In some embodiments, the polynucleotides of the present application consist of nucleotide sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the nucleotide sequence shown in SEQ ID NO:1. In a preferred embodiment, the polynucleotides of the present application consist of the nucleotide sequence shown in SEQ ID NO:1.

[0053] In a specific embodiment, the polynucleotide shown in SEQ ID NO:1 encodes the amino acid sequence shown in SEQ ID NO:2.

[0054] 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 polynucleotide form of nucleotides at least 10 bases in length, where the nucleotides are ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide.

[0055] In certain embodiments, the polynucleotides of the present application comprise nucleotide sequences that hybridize under stringent conditions to a nucleotide sequence encoding the polypeptide shown in SEQ ID NO:2 and its functionally equivalent variants, or consist of nucleotide sequences that specifically hybridize to a nucleotide sequence encoding the polypeptide shown in SEQ ID NO:2 and its functionally equivalent variants and encode a polypeptide that is functionally equivalent to the polypeptide shown in SEQ ID NO:2.

[0056] Those skilled in the art can routinely select the stringent conditions for DNA hybridization. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization usually 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 temperature that can be employed. Thus, higher relative temperatures tend to make the reaction conditions more stringent, while at lower temperatures, the stringency is lower. For a detailed description of the stringent conditions for hybridization reactions, reference can be made to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0057] In certain embodiments, the 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) denaturing agents such as formamide during hybridization, such as 50% (v / v) formamide plus 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 and 750 mM sodium chloride, 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 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 mg / mL), 0.1% SDS, and 10% dextran sulfate, followed by washing in 0.2×SSC (sodium chloride / sodium citrate) at 42 °C for 10 minutes and then high-stringency washing in 0.1×SSC containing EDTA at 55 °C. Moderate stringent conditions can be determined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989. Moderate stringent conditions include using washing solutions and hybridization conditions (such as temperature, ionic strength, and SDS percentage) with a lower stringency than those described above. For example, moderate stringent conditions include hybridization at 42 °C 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, and washing at 55 °C with at least about 0.1 M to at least about 0.2 M salt. Moderate stringent conditions can also include hybridization at 65 °C with 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS, and washing at 60 - 65 °C with (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. A person skilled in the art will adjust the temperature, ionic strength, etc. according to factors such as the probe length. The stringency during hybridization of nucleic acids depends on the length and degree of complementarity of the nucleic acid molecules, 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 these sequences. The relative stability of nucleic acid hybridization (corresponding to a 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.

[0058] The polynucleotides disclosed in the present application can be combined with other DNA sequences, such as promoters, polyadenylation signals, other restriction enzyme cleavage sites, polylinker sites, other coding segments, etc., such that their total lengths can vary significantly. Thus, it is 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.

[0059] Any of a variety of well-established techniques known and available in the art can be used to prepare, manipulate, and / or express the polynucleotides and their fusions. For example, a polynucleotide sequence encoding a polypeptide or a variant thereof of the present application can be used in a recombinant DNA molecule to direct the expression of the polypeptide in a suitable host cell. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can also be used in the present application, and these sequences can be used for cloning and expressing a given polypeptide.

[0060] In addition, the polynucleotide sequences of the present application can be modified using methods well-known in the art, including but not limited to altering the cloning, processing, expression, and / or activity of the gene product.

[0061] 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 above polynucleotides are produced by recombinant techniques.

[0062] In certain embodiments, the sequence of the obtained polynucleotide can be determined by conventional methods, preferably such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74:5463 - 5467). Such polynucleotide sequencing can also be accomplished using commercially available sequencing kits.

[0063] Expression vector

[0064] The present application provides an expression vector comprising the polynucleotide of the present application.

[0065] The "expression vector" described in the present application is a recombinant or synthetically produced nucleic acid construct that has a series of specific nucleic acid elements that permit the transcription of a particular nucleic acid in a host cell. The expression vectors used in the present application can be plasmid vectors such as pPIC9K, pAO815, pUC57, pET - 24a(+), pIRES2 - EGFP, pcDNA3.1, pCI - neo, pDC516, pVAC, pcDNA4.0, pGEM - T, pDC315, or viral vectors such as adenovirus, adeno - associated virus, retrovirus, semliki forest virus (sFv) vectors, or other vectors well-known in the art.

[0066] In certain embodiments, the polynucleotide sequence encoding the polypeptide shown in SEQ ID NO:2 and its variants is cloned into a vector to construct a recombinant vector containing the polynucleotide described in the present application.

[0067] In a preferred embodiment, the expression vector for cloning the polynucleotide is a plasmid vector. In a more preferred embodiment, the plasmid vector is pPIC9K or pAO815.

[0068] 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.

[0069] As used herein, the term "regulatory sequence" refers to the polynucleotide sequence required to effect the expression of the coding sequence to which it is linked. The nature of such regulatory sequences varies with the host organism. In prokaryotes, such regulatory sequences generally include a promoter, a ribosome binding site, and a terminator; in eukaryotes, such regulatory sequences generally include a promoter, a terminator, and in some cases, an enhancer. Thus, the term "regulatory sequence" includes all sequences that are minimally required for the expression of the gene of interest, and may also include other sequences that are beneficial for the expression of the gene of interest, such as a leader sequence.

[0070] As used herein, the term "operably linked" refers to the situation where the sequences involved are in a relationship that allows them to function in the desired manner. Thus, for example, a regulatory sequence "operably linked" to a coding sequence causes the expression of the coding sequence to be achieved under conditions compatible with the regulatory sequence.

[0071] In certain embodiments, an expression vector containing a nucleotide sequence encoding the polypeptide shown in SEQ ID NO:2 and its variants and appropriate transcriptional / translational regulatory elements is constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. (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 promoter of Escherichia coli; the PL promoter of bacteriophage λ; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and some other known promoters that can control gene expression in prokaryotic cells, eukaryotic cells, or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcriptional terminator, etc. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs in length, and act on the promoter to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer, etc.

[0072] 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, etc.

[0073] Host cell

[0074] This application provides host cells containing the polynucleotides or expression vectors disclosed herein.

[0075] In certain embodiments, the polynucleotide encoding the polypeptide shown in SEQ ID NO:2 and its variants or the expression vector containing the polynucleotide is transformed or transfected into a host cell to obtain a genetically engineered host cell containing the polynucleotide or expression vector.

[0076] The host cells used herein can be any host cells 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, etc. Exemplary bacterial cells include any species in the genera Escherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas, and Staphylococcus, including, for example, Escherichia coli, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonas fluorescens. Exemplary fungal cells include any species in the genus Aspergillus. Exemplary yeast cells include any species in the genera Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, including Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe. Exemplary insect cells include any species in 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 a suitable host is within the ability of those skilled in the art.

[0077] In a specific embodiment, the host cell used in the present application is Escherichia coli. In a preferred embodiment, the expression vector carrying the polynucleotide sequence of the present application is transformed into Escherichia coli DH5α strain for induced expression. In another specific embodiment, the expression vector carrying the polynucleotide sequence of the present application is transformed into Pichia pastoris cells for expression. Pichia pastoris that can be used in the present application includes GS115, M314, etc., which are suitable for general yeast transformation methods.

[0078] Any technique known in the art can be used to introduce the expression vector into the host cell, 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, etc. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). As an example, when the host is a prokaryote such as Escherichia coli, competent cells can be harvested after the exponential growth phase and transformed by the well-known electroporation method in the art.

[0079] In a specific embodiment, the recombinant expression vector containing the polynucleotide encoding the TL mutant is digested and linearized with SalI restriction endonuclease, and then transformed into Pichia pastoris competent cells by electroporation. The transformants are inoculated on an MGYS plate for culture. Transformants are picked and cultured on a BMMY-vinyl laurate screening plate, and finally the transformant with the largest hydrolysis zone is picked for testing.

[0080] Application of the polypeptide with lipase activity

[0081] The polypeptide of the present application is a polypeptide with lipase activity, capable of hydrolyzing and / or synthesizing monoglycerides and / or diglycerides.

[0082] When hydrolyzing and / or synthesizing monoglycerides and / or diglycerides, the polypeptide of the present application can be used, or a mixture containing the polypeptide of the present application can be used. For example, but not limited to, fermentation broth, fermentation supernatant, concentrated fermentation broth, etc. containing the polypeptide of the present application.

[0083] In this specification and claims, the words "comprising", "including", and "containing" mean "including but not limited to", and are not intended to exclude other parts, additives, components, or steps.

[0084] It should be understood that the features, characteristics, components, or steps described in a particular aspect, embodiment, or example of the present application can be applied to any other aspect, embodiment, or example described herein, unless there is a contradiction.

[0085] The above disclosure generally describes the present application, and the present application is further exemplified 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.

[0086] Examples

[0087] Experimental materials

[0088] 1) Experimental strains and plasmids

[0089] Plasmid pUC57: Sangon Biotech Co., Ltd.

[0090] Plasmid pPIC9K: Invitrogen

[0091] Pichia pastoris m316H: It was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 19, 2019. The taxonomic name is Pichia pastoris m316H, and its deposit number is CGMCC No. 19221.

[0092] 2) Culture media and solutions

[0093] LB liquid medium: 1% peptone, 0.5% yeast extract, 0.5% sodium chloride.

[0094] LB plate: 1% peptone, 0.5% yeast extract, 0.5% sodium chloride, 1.7% agarose.

[0095] Selective medium MGYS screening plate: 1.34% yeast nitrogen base (YNB), 18.1% sorbitol, 2% glycerol.

[0096] Diolein plate: 1.34% yeast nitrogen base (YNB), 1% yeast extract, 2% peptone, 2% agarose, 2% methanol, 0.5% diolein.

[0097] BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 2% glycerol, 4×10-5% D-biotin, 100 mM citrate-sodium citrate buffer, pH 6.6.

[0098] BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 2% methanol, 4×10-5% D-biotin, 100 mM citrate-sodium citrate buffer, pH 6.6.

[0099] pNPP reaction solution: Dissolved in isopropanol at 3 mg / mL. Take 1 mL and mix it with 9 mL of 0.2 M sodium acetate-acetic acid solution before use, pH 4.8.

[0100] Stop solution: 200 mM Tris-HCL, 5% triton-100, pH 8.5.

[0101] 3) Sequence

[0102] SEQ ID NO.:1 is the nucleotide sequence of the CTLmc mature peptide.

[0103] SEQ ID NO.:2 is the amino acid sequence of CTLmc.

[0104] SEQ ID NO.:3 is the nucleotide sequence of the TL mature peptide.

[0105] SEQ ID NO.:4 is the amino acid sequence of TL.

[0106] Example 1: Construction of the TL mutant library and screening of mutants

[0107] According to the sequence of TL (the sequence is shown as SEQ ID NO.:3, and its coding sequence is shown as SEQ ID NO.:4), the C-terminal sequence from I241 to L269 was artificially designed, and the designed sequence was named CTLmc (the sequence is shown as SEQ ID NO.:1, and its coding sequence is shown as SEQ ID NO.:2). It was sent to Shanghai Sangon Biotech Co., Ltd. for total gene synthesis and ligated by enzyme digestion to the AvrII and EcoRI digestion sites of plasmid pUC57 to obtain pUC57-CTLmc.

[0108] The plasmid pUC57-CTLmc was digested with AvrII and EcoRI from NEB. Meanwhile, the commercial plasmid pPIC9K with the inducible promoter PAOX1 and yeast α-mating factor was digested with AvrII and EcoRI, and then purified using the gel extraction kit from Axygen. Using the T4 DNA ligase from Fermentas and following the product instructions, the digested fragment of the CTLmc sequence and the digested plasmid fragment were ligated. The ligation product was transformed into Escherichia coli DH5α by heat shock and cultured overnight on an LB plate containing ampicillin. The next day, single colonies were picked and cultured in LB liquid medium. The plasmid was extracted using the plasmid extraction kit from Axygen and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain a correctly sequenced recombinant expression vector.

[0109] Example 2: Transformation of CTLmc into Pichia pastoris and plate screening

[0110] The correctly sequenced recombinant expression vector was digested and linearized with the SalI restriction endonuclease. Competent cells of m316H were prepared referring to the Pichia pastoris operation manual (Invitrogen), and then the linearized plasmid was electrotransformed into the competent cells of Pichia pastoris m316H. The transformants were spread on the selection medium MGYS screening plate and cultured at 28 °C for three days. The transformants were picked onto the diolein plate and cultured at 28 °C for 1 day.

[0111] Example 3: Fermentation test of CTLmc-producing strains

[0112] Three monoclonal colonies with larger clear zones on the diolein plate were picked, named CM1, CM2, and CM3 respectively. The three strains were respectively inoculated into 50 ml of BMGY medium and cultured at 28 °C and 200 rpm for 1 day until the OD600 was about 10. A culture solution with a total cell mass of 300 OD was taken, centrifuged at 8000 rpm for 2 minutes, and resuspended in a 250 ml Erlenmeyer flask containing 50 ml of BMMY. The initial OD600 of the bacterial solution was 5, and it was cultured at 28 °C and 200 rpm for 3 days. Methanol (500 μl) was added every 12 h. After the culture was completed, all the bacterial solutions were collected, centrifuged at 8000 rpm for 5 minutes, and all the supernatants were taken and ultrafiltered using a 10K filter membrane (purchased from Millipore) to form an enzyme solution for protein concentration determination, enzyme activity determination, polyacrylamide gel electrophoresis analysis, and hydrolysis test. The protein concentration was determined using Bradford reagent (purchased from Sangon Biotech Co., Ltd.). The protein concentration in the fermentation broth was 0.20 - 0.45 mg / ml, and the results are as Figure 1 shown.

[0113] Example 4: Detection of MDGL hydrolysis activity by pNPP method

[0114] Take 400 μl of pNPP reaction solution, add 5 μl of enzyme solution, react at 37 °C and 1000 rpm for 20 minutes, then add 400 μl of termination solution to terminate the reaction. Centrifuge at 12000 rpm for 5 min, take the supernatant, measure the absorbance at 405 nm, and the enzyme activity unit detection method is as follows:

[0115] Enzyme activity unit = 0.1935 * OD * V1 * dilution factor / T / V2, where V1 is the volume of the reaction solution (ml), V2 is the volume of the enzyme (ml), and T is the time (min);

[0116] After detection, the highest enzyme activity yield of CTLmc is 2.0 U / ml( Figure 2 ).

[0117] Example 5: Test of CTLmc on the hydrolysis of mono- and dioleoyl glycerols

[0118] Perform a test on the hydrolysis of mono- and dioleoyl glycerols by CTLmc (react at 37 °C and 1000 rpm for 2 h on an eppendorf mixer), and the reaction system is shown in Table 1.

[0119] Table 1

[0120] Component Dosage Monolein or diolein 10ul <![CDATA[ddH2O]]> 9ul Enzyme solution / water 1ul Total volume 20 μl

[0121] After the reaction, add 250 μl of n-hexane, mix well and centrifuge at 12000 rpm for 10 min. Take 200 μl of the supernatant and evaporate it to dryness. Add 50 μl of n-hexane to dissolve the oil sample, and take 2 μl to load onto a thin-layer chromatography plate (Merck). The developing agent is n-hexane:ethyl ether:ethyl acetate:acetic acid = 29:19:1:0.5, and iodine fuming is carried out for about 5 min. Among them, the results of using the enzyme solution of CM1 are as Figure 3 shown (the results of CM2 and CM3 are similar and not shown).

[0122] According to Figure 3 the results, different from TL which can only hydrolyze trioleoyl glycerol and dioleoyl glycerol, CTLmc can hydrolyze monooleoyl glycerol into oleic acid and glycerol, and can hydrolyze dioleoyl glycerol into oleic acid and glycerol, and the ability to hydrolyze monooleoyl glycerol is stronger than that to hydrolyze dioleoyl glycerol.

[0123] It can be understood that although this application is described in a certain form, this application is not limited to the content shown and described in this specification. It is obvious to those skilled in the art that various changes can be made without departing from the scope of this application. These changes are all within the scope claimed by this application. Sequence Listing <110> Wilmar (Shanghai) Biotechnology R & D Center Co., Ltd. <120> A novel mono-diacylglycerol lipase <130> 123234 <160> 4 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 807 <212> DNA <213> Artificial Sequence () <400> 1 gtctctcaag acttgttcaa ccagttcaac ttgttcgctc aatactctgc cgctgcctac 60 tgtggtaaga acaatgatgc tccagctggt actaacatta cctgtactgg taacgcttgt 120 ccagaagttg agaaggctga tgctaccttc ctgtactcct tcgaagactc tggagttgga 180 gatgttactg gtttcctggc cttggataac actaacaagt tgatcgttct gtccttcaga 240 ggttccagat ccatcgagaa ctggattggt aacttgaact ttgacttgaa ggagatcaac 300 gacatctgtt ctggatgtcg tggtcacgat ggatttacct cctcttggag atctgttgct 360 gataccttga gacagaaggt cgaagatgct gtcagagaac atccagacta tagagttgtc 420 ttcactggtc actccttggg aggtgccttg gctactgttg ctggtgctga cttgcgtggt 480 aatggttatg acattgatgt cttctcctac ggtgctccaa gagttggtaa tcgtgccttc 540 <h2 style=";text-align:left;direction:ltr">gctgagtttc tgaccgtcca aactggaggt actttgtaca gaattaccca tactaacgac 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> attgttccaa gattgccacc acgtgagttc ggatactctc attcctctcc agagtactgg 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atcaagtctg gaaccttggt tccagtcaga cgtagagaca tcgtcaagat tgaaggtttt 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gacggcaata ccggcacggg cctgcctctg ctgacggact ttgaagccca catttggtac 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttgtacagg ttgacgccgg caaataa 807<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 269<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213>人工序列()<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50 55 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 65 70 75 80<h2 style=";text-align:left;direction:ltr"> 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 Arg Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Phe Asp Gly Asn Thr Gly Thr Gly Leu Pro Leu Leu Thr Asp Phe Glu 245 250 255 Ala His Ile Trp Tyr Phe Val Gln Val Asp Ala Gly Lys 260 265 <210> 3 <211> 807 <212> DNA <213> Thermomyces lanuginosus <400> 3 gtctctcaag acttgttcaa ccagttcaac ttgttcgctc aatactctgc cgctgcctac 60 tgtggtaaga acaatgatgc tccagctggt actaacatta cctgtactgg taacgcttgt 120 ccagaagttg agaaggctga tgctaccttc ctgtactcct tcgaagactc tggagttgga 180 gatgttactg gtttcctggc cttggataac actaacaagt tgatcgttct gtccttcaga 240 ggttccagat ccatcgagaa ctggattggt aacttgaact ttgacttgaa ggagatcaac 300 gacatctgtt ctggatgtcg tggtcacgat ggatttacct cctcttggag atctgttgct 360 gataccttga gacagaaggt cgaagatgct gtcagagaac atccagacta tagagttgtc 420 ttcactggtc actccttggg aggtgccttg gctactgttg ctggtgctga cttgcgtggt 480 aatggttatg acattgatgt cttctcctac ggtgctccaa gagttggtaa tcgtgccttc 540 gctgagtttc tgaccgtcca aactggaggt actttgtaca gaattaccca tactaacgac 600 attgttccaa gattgccacc acgtgagttc ggatactctc attcctctcc agagtactgg 660 atcaagtctg gaaccttggt tccagtcaga cgtagagaca tcgtcaagat tgaaggtatt 720 gatgccactg gaggtaacaa tcaaccaaac attccagaca ttccagctca cttgtggtac 780 tttggtctga ttggtacttg cttgtaa 807 <210> 4 <211> 269 <212> PRT <213> Thermomyces lanuginosus <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 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 Arg 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

Claims

1. A polypeptide having lipase activity, which consists of: the amino acid sequence shown in SEQ ID NO:

2.

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

3. The polynucleotide according to claim 2, which consists of the nucleotide sequence shown in SEQ ID NO:

1.

4. An expression vector comprising at least one 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. A host cell comprising the polynucleotide according to claim 2 or 3, or the expression vector according to claim 4 or 5, wherein the host cell is not a plant cell.

7. The host cell according to claim 6 is yeast.

8. Use of the polypeptide according to claim 1 for the hydrolysis of monoglycerides and / or diglycerides.

9. A method for hydrolyzing monoglycerides and / or diglycerides, characterized in that, The method uses the polypeptide according to claim 1 or the fermentation broth or supernatant or concentrate of the host cell according to claim 6 or 7 to catalyze the monoglycerides and / or diglycerides.

Images