CN109929820B - Novel glycerol mono-diacyl ester lipase - Google Patents

Abstract

The present application provides novel polypeptides having glycerol mono-diacylate lipase activity comprising the amino acid sequence shown as SEQ ID No.1 or a sequence comprising at least one amino acid substitution, deletion or addition in the above sequence. The application also provides polynucleotides encoding the polypeptides, expression vectors and host cells comprising the polynucleotides. In addition, the application also relates to the application of the polypeptide with lipase activity.

Description

Novel glycerol mono-diacyl ester lipase

Technical Field

The present application is in the field of enzyme engineering, and in particular, relates to polypeptides having lipase activity, nucleic acids encoding the same, and expression vectors and host cells comprising the encoding nucleic acids. The application also relates to a screening method of the polypeptide and application thereof.

Background

The lipase is one of esterases, and can catalyze the hydrolysis of triglyceride, diglyceride, monoglyceride, other small molecular esters, polyol ester and ester bonds of the polybasic acid ester, fat is a natural substrate of the lipase, the diglyceride and the monoglyceride are generated in the hydrolysis process, and the final products of the hydrolysis are glycerol and fatty acid. The lipase has mild hydrolysis condition, less by-products and no need of coenzyme. Most fats are hydrophobic, and thus hydrolysis occurs at the oil-water interface or in the organic phase. Glycerol mono-diacyl ester lipase (MDGL) is one of lipases, and MDGL has substrate specificity, acts only on glycerol monoacyl ester (MAG) and glycerol diacyl ester (DAG), does not catalyze glycerol triacyl ester, and can produce glycerol monoacyl ester with high industrial value by using esterification or transesterification.

MAG is an excellent emulsifier and has wide applications in the food, pharmaceutical and cosmetic industries. The traditional synthesis process of MAG is to produce MAG by continuous esterification of grease/Triglyceride (TAG) and glycerol at high temperature and under the condition of nitrogen by taking inorganic base as a catalyst, wherein the product is a mixture of MAG, DAG and TAG, and MAG is obtained by distillation, and the yield of MAG is 40-50%. Different from the traditional synthesis process, the enzymatic synthesis of MAG takes fatty acid and glycerol as substrates, and the MAG is synthesized by catalysis under mild conditions, wherein the conversion rate of the fatty acid in the enzymatic catalysis is over 97 percent, and the proportion of MAG in the product is over 74 percent. The major problems currently limiting the use of enzymatic methods are the low enzymatic activity and the high cost of the enzyme.

Summary of The Invention

In a first aspect, there is provided a polypeptide having glycerol mono-diacylate lipase activity comprising or consisting of a sequence selected from:

(a) 1, and an amino acid sequence as shown in SEQ ID NO, and

(b) A sequence obtained by substituting, deleting or adding at least one amino acid to the sequence of (a), wherein the polypeptide variant obtained from (b) still maintains glycerol mono-diacylate lipase activity.

In one embodiment, the polypeptide of the present application comprises the amino acid sequence set forth in SEQ ID NO. 1. In a preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO. 1. Herein, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1 is designated as MDGL-new.

In a second aspect, there is provided a polynucleotide encoding a polypeptide of the first aspect comprising or consisting of a sequence selected from:

(a) A nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO.1 or a sequence comprising at least one amino acid substitution, deletion or addition in the above sequence; and

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

In one embodiment, the polynucleotide of the present application comprises the nucleotide sequence set forth in SEQ ID NO. 2. In a preferred embodiment, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO. 2.

In a third aspect, there is provided an expression vector comprising at least one polynucleotide of the second aspect.

In certain embodiments, the expression vectors of the present application further comprise a control sequence that regulates expression of the polynucleotide, wherein the polynucleotide is operably linked to the control sequence. In a preferred embodiment, the expression vector is a plasmid vector, such as P0586-G50.

In a fourth aspect, there is provided a host cell comprising the polynucleotide of the second aspect or the expression vector of the third aspect. In specific embodiments, the host cell is a yeast such as pichia, or escherichia coli.

In a fifth aspect, there is provided the use of a polypeptide of the first aspect in the preparation of a monoglyceride.

In a sixth aspect, there is provided a method of synthesizing a monoacyl glyceride comprising contacting a polypeptide of the first aspect with a fatty acid and glycerol.

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

1) Randomly mutating the nucleotide sequence shown in SEQ ID NO. 3 to obtain a mutation sequence set,

2) Cloning the mutant sequence obtained in step 1) on an expression vector, then transforming or transducing the mutant sequence into a suitable host cell to obtain a mutant library,

3) Culturing the host cell of step 2), recovering the recombinant expression vector, and then transforming the linearized product into a yeast strain for culturing, and

4) Screening the mutant polypeptide with enzyme activity higher than that of the polypeptide shown in SEQ ID NO. 4.

In addition, the application also provides the use of the nucleic acid sequence shown in SEQ ID NO. 3 for screening the polypeptides disclosed herein.

The polypeptides obtained by the screening methods disclosed herein have high lipase activity, particularly glycerol mono-diacylate lipase activity.

Brief description of the drawings

FIG. 1 shows the protein concentration and enzyme activity of MDGL expressed in Pichia pastoris GS 115.5L fermentor, where MDGL enzyme activity was determined at different fermentation time based on PNPP method.

FIG. 2 shows the results of SDS-PAGE for the commercial enzymes MDGL and MDGL-new, wherein "M" represents a molecular marker, "A" represents the commercial enzyme Amano G50, and "W1", "W2" and "W3" represent MDGL-new.

Brief description of the sequences

1, SEQ ID NO: the amino acid sequence of MDGL-new;

2, SEQ ID NO: a nucleic acid sequence encoding MDGL-new;

3, SEQ ID NO: an optimized MDGL nucleic acid sequence;

4, SEQ ID NO: optimized MDGL amino acid sequence;

5, SEQ ID NO: a forward amplification primer of the optimized MDGL;

6 of SEQ ID NO: optimized reverse amplification primer for MDGL:

7, SEQ ID NO: forward amplification primers for MDGL-new;

8, SEQ ID NO: a reverse amplification primer of MDGL-new;

9 of SEQ ID NO: forward amplification primers for MDGL-new;

10, SEQ ID NO: MDGL-new reverse amplification primer.

Detailed Description

Polypeptides of the present application

The present application provides polypeptides having lipase activity, in particular glycerol mono-diacylate lipase activity, comprising or consisting of a sequence selected from:

(a) 1, and an amino acid sequence as shown in SEQ ID NO, and

(b) A sequence obtained by substituting, deleting or adding at least one amino acid to the sequence of (a), wherein the polypeptide variant obtained in (b) still maintains lipase activity.

Glycerol mono-diacyl ester lipase (MDGL) is one type of lipase. MDGL has 2 forms, and has molecular weight of 37kDa and 39kDa, and has 26 amino acids as signal peptide, and mature peptide contains 279 amino acids. For example, the MDGL enzyme activity units can be determined using vinyl laurate as a substrate.

In certain embodiments, the number of such amino acid substitutions, deletions or additions is from 1 to 30, preferably from 1 to 20, more preferably from 1 to 10, wherein the resulting polypeptide variant substantially retains the lipase activity of the unaltered protein. In preferred embodiments, the above polypeptide variants differ from the amino acid sequence shown in SEQ ID No.1 by substitutions, deletions and/or additions of about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In a more preferred embodiment, the above polypeptide variant differs from the amino acid sequence shown in SEQ ID NO.1 by substitutions, deletions or additions of about 1, 2,3, 4 or 5 amino acids.

In one embodiment, the polypeptide of the present application comprises the amino acid sequence shown in SEQ ID NO. 1. In a preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO. 1. Herein, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1 is designated as MDGL-new.

The sequence of SEQ ID NO.1 of the present application is shown below:

DVSTSELDQFEFWVQYAAASYYEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFSDSTITDTAGYIAVDHTNSAVVLAFRGSHSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKELKEVVAQNPDYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAYASPRVGNAALAKYITAQGNNFRFTHTNDPVPKLPLLSMGYVHVSPEYWITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAGKGPGLPFKRV

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, ethylmethionine, and the like, as known to those skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications may include, for example, substitution of chemical groups and moieties on the amino acids, or derivatization of 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 an amino acid. For example, the organic structure that mimics arginine (Arg or R) has a positively charged moiety 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 constraining structures to maintain optimal steric and charge interactions of amino acid or amino acid functional groups. One skilled in the art can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

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

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

The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues and variants 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 shown in SEQ ID NO: 1.

In a preferred embodiment, the sequence of the polypeptide variant is a sequence comprising one or several conservative amino acid substitutions in the amino acid sequence shown in SEQ ID NO.1, wherein the substituted sequence still retains a similar lipase catalytic activity, in particular glycerol mono-diacylate lipase activity.

Certain amino acid substitutions, known as "conservative amino acid substitutions," can occur frequently in proteins without changing the conformation or function of the protein, a well-established rule in protein chemistry.

Conservative amino acid substitutions in this application include, but are not limited to, substitution of any one of these aliphatic amino acids with any one of glycine (G), alanine (a), isoleucine (I), valine (V), and leucine (L); substitution of threonine (T) with serine (S) and vice versa; substitution of aspartic acid (D) for glutamic acid (E), and vice versa; substitution of asparagine (N) with glutamine (Q), and vice versa; substitution of arginine (R) with lysine (K) and vice versa; substitution of any one of these aromatic amino acids with phenylalanine (F), tyrosine (Y) and tryptophan (W); and substitution of 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) are often interchangeable, as are alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged for leucine and isoleucine, and sometimes for valine. Lysine (K) and arginine (R) are often interchanged at the following positions: the important characteristics of the amino acid residues are their charge and the different pKs of the two amino acid residues are not significant. Still other variations may be considered "conservative" under certain circumstances (see, e.g., BIOC)HEMISTRY at pp.13-15,2 ^nd ed.Lubert Stryer ed.(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)。

In the following, amino acid residues are exemplified by the group of substitutable residues, but the substitutable amino acid residues are not limited to the residues described below:

group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-methylserine, tert-butylglycine and cyclohexylalanine;

group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid and 2-aminosuberic acid;

group C: asparagine and glutamine;

group D: lysine, arginine, ornithine, 2, 4-diaminobutyric acid, i.e., 2, 3-diaminopropionic acid;

group E: proline, 3-hydroxyproline and 4-hydroxyproline;

and F group: serine, threonine, and homoserine;

group G: phenylalanine, and tyrosine.

In certain embodiments, a polypeptide of the present application, e.g., a polypeptide as set forth in SEQ ID NO.1 or a variant thereof, is fused to a heterologous polypeptide. In some embodiments, the fusion protein substantially retains the lipase activity of the polypeptide set forth in SEQ ID NO. 1. In certain embodiments, the heterologous polypeptide is linked to the N-terminus of the polypeptide set forth in SEQ ID NO. 1. In certain embodiments, the heterologous polypeptide is linked to the C-terminus of the polypeptide set forth in SEQ ID NO. 1. In these embodiments, the heterologous polypeptide can be selected from a purification tag (e.g., can include, but is not limited to: GST, MBP), an epitope tag (e.g., can include, but is not limited to: myc, FLAG), a targeting sequence, a signal peptide, and the like.

In a particular embodiment, the fusion protein comprises a polypeptide of SEQ ID NO.1 and a tag, typically a peptide tag, attached to the C-terminus or N-terminus of the polypeptide of SEQ ID NO. 1. The tag is typically a peptide or amino acid sequence that can be used to isolate and purify the fusion protein.

Polynucleotide

The present application provides polynucleotides encoding the polypeptides disclosed herein comprising or consisting of a sequence selected from:

(a) A nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO.1 or a sequence comprising at least one amino acid substitution, deletion or addition in the above sequence; and

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

In certain specific embodiments, the polynucleotides of the present application encode the polypeptide set forth in SEQ ID NO.1 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 to polynucleotides encoding the polypeptide set forth in SEQ ID NO.1 and functionally equivalent variants thereof.

In certain embodiments, the polynucleotides of the present application comprise a nucleotide sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the nucleotide sequence set forth in SEQ ID NO. 2. In a preferred embodiment, the polynucleotide of the present application comprises the nucleotide sequence shown in SEQ ID NO. 2.

The sequence of SEQ ID NO 2 is shown below:

GATGTCTCCACTTCCGAACTGGACCAGTTCGAGTTCTGGGTACAATACGCAGCCGCCTCTTACTACGAGGCTGATTACACCGCACAGGTTGGTGATAAGCTGTCCTGCTCTAAGGGTAACTGCCCAGAAGTTGAAGCAACCGGTGCAACTGTGTCTTAC GACTTCTCCGATTCCACGATCACTGACACCGCAGGTTACATCGCAGTTGATCACACCAACTCCGCAGTGGTACTGGCATTCCGTGGTTCTCACTCCGTACGTAACTGGGTTGCTGATGCTACTTTCGTCCATACCAACCCAGGTCTGTGTGATGGTTGCCTGGCTGAGCTGGGTTTCTGGTCTTCCTGGAAGCTGGTTCGTGATGATATTATCAAAGAACTGAAAGAAGTGGTGGCACAGAACCCAGACTATGAACTGGTGGTCGTGGGCCACTCCCTGGGTGCTGCTGTGGCTACTCTGGCTGCTACCGACCTGCGTGGTAAAGGTTATCCATCTGCTAAACTGTACGCCTACGCTTCCCCTCGTGTTGGCAACGCAGCCCTGGCCAAATATATCACCGCCCAGGGCAACAACTTCCGTTTCACCCACACCAATGACCCAGTACCTAAACTGCCACTGCTGTCTATGGGCTATGTACATGTTTCTCCTGAATATTGGATCACCTCTCCTAACAACGCCACTGTTTCTACCTCTGACATCAAAGTCATTGACGGCGACGTATCTTTTGACGGCAATACCGGCACGGGCCTGCCTCTGCTGACGGACTTTGAAGCCCACATTTGGTACTTTGTACAGGTTGACGCCGGCAAAGGTCCTGGCCTGCCATTCAAACGTGTTTAA

in preferred 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 with the nucleotide sequence depicted in SEQ ID NO. 2. In a more preferred embodiment, the polynucleotide of the present application consists of the nucleotide sequence shown in SEQ ID NO. 2.

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

In certain embodiments, the polynucleotides of the present application comprise or consist of a nucleotide sequence that hybridizes specifically with a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO.1 and functionally equivalent variants thereof under stringent conditions and that encodes a polypeptide functionally equivalent to the polypeptide set forth in SEQ ID NO. 1.

Stringent conditions for DNA hybridization can be routinely selected by those skilled in the art. 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 strand is in an environment below its melting temperature. The higher the degree of homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. 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, see Ausubel et al, current Protocols in Molecular Biology, wiley Interscience Publishers (1995).

In certain embodiments, the stringent conditions employed for DNA hybridization include: 1) Washing with low ionic strength and high temperature, e.g. 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate at 50 ℃; 2) Hybridization is carried out using a denaturing agent such as formamide, e.g., 50% (v/v) formamide plus 0.1% bovine serum albumin/0.1% Ficoll/0.1% polydiallylpyrrolidone/50 mM sodium phosphate buffer pH 6.5 at 42 ℃ with 750mM sodium chloride, 75mM sodium citrate; or (3) overnight hybridization at 42 ℃ in a hybridization solution containing 50% formamide, 5 XSSC (0.75M sodium chloride, 0.075M rotten 1 sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5XDenhardt's solution, sonicated salmon sperm DNA (50.mu.g/ml), 0.1% SDS and 10% dextran sulfate, followed by washing in 0.2 XSSC (sodium chloride/sodium citrate) at 42 ℃ for 10 minutes and high stringency washing in 0.1 XSSC with EDTA at 55 ℃. Moderately stringent conditions can be determined by Sambrook et al, molecular Cloning: a Laboratory Manual, new York: determined as described in Cold Spring Harbor Press, 1989. Moderately stringent conditions include those that employ wash solutions and hybridization conditions (e.g., temperature, ionic strength, and percentage SDS) that are less stringent than those described above. For example, moderately stringent conditions comprise hybridization at 42 ℃ with at least about 16% v/v to at least about 30% v/v of formamide and at least about 0.5M to at least about 0.9M salt, and washing at 55 ℃ with at least about 0.1M to at least about 0.2M salt. Moderately stringent conditions may further comprise hybridization at 65 ℃ with 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5M NaHPO4 (pH 7.2), 7% SDS, and (i) 2 XSSC, 0.1% SDS; or (ii) 0.5% BSA, 1mM EDTA, 40mM NaHPO4 (pH 47.2), 5% SDS washed at 60-65 ℃. The practitioner will adjust the temperature, ionic strength, etc. depending on factors such as probe length. The stringency of hybridization of 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 for hybrids of nucleic acids containing those sequences. The relative stability of nucleic acid hybridization (corresponding to higher Tm) decreases in the following order: RNA, DNA, RNA, DNA. Preferably, the minimum length of the hybridizable nucleic acid 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.

In some embodiments, the inventors of the present application performed codon optimization of the sequence encoding the mature protein based on mdlA gene from Penicilliurn camembertii U-150 according to the codon preference of pichia pastoris, and the synthesized gene was expressed with pichia pastoris GS115 as an expression host.

In a specific embodiment, the present application provides a codon-optimized nucleotide sequence (SEQ ID NO: 3) encoding the mature peptide of glycerol mono-diacyl lipase (SEQ ID NO: 4).

The sequence of SEQ ID NO 3 is shown below:

GATGTCTCCACTTCCGAACTGGACCAGTTCGAGTTCTGGGTTCAATACGCAGCCGCCTCTTACTACGAGGCTGATTACACCGCACAGGTTGGTGATAAGCTGTCCTGCTCTAAGGGTAACTGCCCAGAAGTTGAAGCAACCGGTGCAACTGTGTCTTACGACTTCTCCGATTCCACGATCACTGACACCGCAGGTTACATCGCAGTTGATCACACCAACTCCGCAGTGGTACTGGCATTCCGTGGTTCTTACTCCGTACGTAACTGGGTTGCTGATGCTACTTTCGTCCATACCAACCCAGGTCTGTGTGATGGTTGTCTGGCTGAGCTGGGTTTCTGGTCTTCCTGGAAGCTGGTTCGTGATGATATTATCAAAGAACTGAAAGAAGTGGTGGCACAGAACCCAAACTATGAACTGGTGGTCGTGGGCCACTCCCTGGGTGCTGCTGTGGCTACTCTGGCTGCTACCGACCTGCGTGGTAAAGGTTATCCATCTGCTAAACTGTACGCTTACGCTTCCCCTCGTGTTGGCAACGCAGCCCTGGCCAAATATATCACCGCCCAGGGCAACAACTTCCGTTTCACCCACACCAATGACCCAGTACCTAAACTGCCACTGCTGTCTATGGGCTATGTACATGTTTCTCCTGAATATTGGATCACCTCTCCTAACAACGCCACTGTTTCTACCTCTGACATCAAAGTCATTGACGGCGACGTATCTTTTGACGGCAATACCGGCACGGGCCTGCCTCTGCTGACGGACTTTGAAGCCCACATTTGGTACTTTGTACAGGTTGACGCCGGCAAAGGTCCTGGCCTGCCATTCAAACGTGTTTAA

the sequence of SEQ ID NO 4 is shown below:

DVSTSELDQFEFWVQYAAASYYEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFSDSTITDTAGYIAVDHTNSAVVLAFRGSYSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAYASPRVGNAALAKYITAQGNNFRFTHTNDPVPKLPLLSMGYVHVSPEYWITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAGKGPGL PFKRV

the polynucleotides disclosed herein may be combined with other DNA sequences, such as promoters, polyadenylation signals, other restriction sites, multiple cloning sites, other coding segments, and the like, such that their overall lengths may vary significantly. It is therefore contemplated that polynucleotide fragments of almost any length may be utilized; the overall length is preferably limited by the ease of preparation and use in contemplated recombinant DNA protocols.

Polynucleotides and fusions thereof can be prepared, manipulated, and/or expressed using any of a variety of mature techniques known and available in the art. For example, a polynucleotide sequence encoding a polypeptide of the present application or a variant thereof may be used in a recombinant DNA molecule to direct expression of the polypeptide in an appropriate host cell. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences may also be used in the present application, and these sequences may be used to clone and express a given polypeptide.

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

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 described above are produced by recombinant techniques.

In a specific embodiment, the MDGL-encoding gene mdlA used herein is derived from Penicilliur camembertii U-150 (genebank number: BAA 14345.1), codons are optimized according to the codon preference of Pichia pastoris, the optimized gene is synthesized by Biotechnology engineering, inc., and the synthesized gene is ligated into pUC57.

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

Expression vector

The present application provides expression vectors comprising the polynucleotides of the present application.

An "expression vector" as described herein is a nucleic acid construct, produced recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector used herein may be a plasmid vector such as P0586-G50, pUC57, pET-24a (+), pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315, or a viral vector such as an adenovirus, adeno-associated virus, retrovirus, semliki forest virus (sFv) vector, or other vectors well known in the art.

In certain embodiments, polynucleotide sequences encoding the polypeptide set forth in SEQ ID NO.1 and variants thereof are cloned into vectors to form recombinant vectors comprising the polynucleotides described herein.

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 P0586-G50 or pUC57.

In a specific embodiment, the above-described expression vector further comprises a control sequence that regulates expression of a polynucleotide, wherein the polynucleotide is operably linked to the control sequence.

The term "control sequences" as used herein refers to polynucleotide sequences required to effect expression of a coding sequence to which they are ligated. The nature of such regulatory sequences varies with the host organism. In prokaryotes, such regulatory sequences typically include a promoter, a ribosome binding site, and a terminator; 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.

The term "operably linked" as used herein refers to the situation wherein: 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 is such that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

In certain embodiments, expression vectors comprising a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO.1 and variants thereof and suitable transcription/translation 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 recombinant techniques, etc. (Sambrook, 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 an expression vector to direct mRNA synthesis. Representative examples of such promoters include: lac or trp promoter of E.coli; the PL promoter of lambda phage; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters which can control the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector may further include a ribosome binding site for translation initiation, a transcription terminator, and the like. The insertion of enhancer sequences into vectors will enhance transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase gene transcription. Examples include the SV40 enhancer, 100 to 270 bp on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers, among others.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli, and the like.

Host cell

The present application provides host cells comprising a polynucleotide or expression vector disclosed herein.

In certain embodiments, a polynucleotide encoding a polypeptide set forth in SEQ ID No.1 and variants thereof, or an expression vector comprising the polynucleotide, is transformed or transduced into a host cell to obtain a genetically engineered host cell comprising the polynucleotide or expression vector.

The host cell used herein may be any host cell 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, and the like. Exemplary bacterial cells include any of 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 of Aspergillus. Exemplary yeast cells include any of the genera Pichia, saccharomyces, schizosaccharomyces, or Saccharomyces, including Pichia, saccharomyces, or Schizosaccharomyces. Exemplary insect cells include spodoptera litura or any of the species drosophila, including drosophila S2 and spodoptera Sf9. Exemplary animal cells include CHO, COS or melanoma or any mouse or human cell line. The selection of a suitable host is within the ability of those skilled in the art.

In a particular embodiment, the host cell used herein is E.coli. In a preferred embodiment, expression vectors carrying the polynucleotide sequences of the present application are transformed into E.coli strain DH 5. Alpha. For inducible expression. In another specific embodiment, an expression vector carrying a polynucleotide sequence of the present application is transformed into a pichia cell for expression. Pichia pastoris that can be used in the present application include both GS115 and KM71, both with HIS4 auxotrophic markers. The GS115 strain has an AOX1 gene and is Mut +, namely a methanol utilization normal type; whereas, the AOX1 site of KM71 strain was inserted with ARG4 gene and the phenotype was Muts, i.e., methanol utilization slow type, both strains were suitable for general yeast transformation methods.

The expression vector may 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. In particular toIncluding calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection, or electroporation, among others (Davis, L., dibner, M., battey, I., basic Methods in Molecular Biology, (1986)). By way of example, when the host is prokaryotic, such as E.coli, the competent cells may be harvested after exponential growth phase, using CaCl as is well known in the art ₂ The method is used for transformation.

In a specific embodiment, a sequence of the MDGL connected with a plasmid pUC57 and coded by the application is cut by AvrII and EcoRI, the MDGL is connected to a pPIC9K (cut by AvrII and EcoRI) vector, the connected vector is transferred into escherichia coli, a single clone is selected for colony PCR verification, positive strain sequencing verification is carried out, a strain with a correct sequencing result is used for extracting the plasmid, the plasmid is converted into pichia pastoris by an electric shock method after being linearized by BglII, a transformant is screened on a screening culture medium without histidine, and only recombinant pichia pastoris transformed with an exogenous gene fragment can grow on the screening culture medium. Transferring the bacterial colony on the screened culture medium to a BMGY-glycerol mono-diacyl ester screening culture medium plate, selecting clone according to the size of a hydrolysis ring, performing shake flask fermentation and activity detection to obtain recombinant pichia pastoris-MDGL, and performing fermentation detection on protein expression capacity and enzyme activity by using a fermentation tank.

Methods for screening and preparing polypeptides of the present application

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

In some embodiments, the polypeptides disclosed herein are obtained by error-prone PCR and mass screening. The MDGL polypeptide can be obtained through random screening, and the result can be verified, so that the MDGL polypeptide can also be used for guiding the directional modification of MDGL.

In some embodiments, the polypeptides of the present application may also be produced by recombinant techniques, or chemically synthesized. Methods of producing recombinant peptides are known in the art. Chemical synthesis methods for peptides are also well known to those skilled in the art, e.g., the polypeptides of the present application and variants thereof can be produced by directed peptide synthesis using solid phase techniques (Merrifield, J.Am.chem.Soc.85:2149-2154 (1963)). Protein synthesis can be performed manually or by automation. Automated synthesis can be achieved, for example, using a 431A peptide synthesizer (Perkin Elmer) from Applied Biosystems. Alternatively, the different degrees of fragmentation can be separately chemically synthesized and chemically combined to produce the desired molecule.

In a specific embodiment, random mutagenesis of the mdlA gene is performed using an error-prone PCR method, and error-prone PCR is performed using TaKaRa Taq enzyme and primers to yield a set of mutant amplicon fragments, e.g., fragments of about 1000bp in size. The resulting fragment was cloned into a plasmid vector such as P0586-G50 by Sac-II and EcoRI enzymatic cleavage sites, and the resulting vector was transformed into a host cell such as E.coli to obtain a mutant library. After the mutant is cultured in, for example, a medium containing ampicillin, the plasmid is extracted, linearized with SalI, and the amplified recombinant vector (for example, a fragment of about 8.5 kb) is recovered and introduced into yeast cells. And culturing the yeast cells introduced with the recombinant vector on a screening culture medium plate, and screening the mutant with high specific enzyme activity by measuring the enzyme activity of a supernatant culture medium.

In some embodiments, the screened mutants may also be validated, for example, fermentation validated. In some embodiments, the validated mutants are sequenced and the mutated sequences are determined.

In some embodiments, the mutated sequence obtained by screening is cloned into a vector, such as a plasmid vector, and then introduced into a yeast cell, for example, the vector is transformed into a competent cell of pichia pastoris GS115 strain by an electric transformation method, and the specific enzyme activity of the polypeptide is verified by fermentation.

In some embodiments, the methods of screening for polypeptides disclosed herein comprise:

1) Randomly mutating the nucleotide sequence shown in SEQ ID NO. 3 to obtain a mutation sequence set,

2) Cloning the mutant sequence obtained in step 1) on an expression vector, then transforming or transducing the mutant sequence into a suitable host cell to obtain a mutant library,

3) Culturing the host cell of step 2) in a suitable medium, recovering the recombinant expression vector, then transforming the linearized product into a yeast strain for culturing, and

4) Screening mutant polypeptide with enzyme activity higher than that of the polypeptide shown in SEQ ID NO. 4.

Suitable host cells refer to host cells suitable for expression of the expression vector or polynucleotide of interest. Suitable medium means a medium suitable for growth of the host cell or for inducible expression thereof.

In certain embodiments, various conventional media may be selected depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. Preferably, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating the promoter to screen for transformants or to amplify the polynucleotides of the present application. After transformation of a suitable host cell and growth of the host cell to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction), and the cell is cultured for an additional period of time to allow production of the polypeptide of interest or a fragment thereof.

In certain embodiments, the polypeptide produced by the host cell may be encapsulated within the cell, or expressed on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. For example, the expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell culture by the following methods well known in the art: conventional renaturation treatment, protein precipitant treatment (salting-out method), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

In particular embodiments, the polypeptides disclosed herein are secreted outside the host cell, e.g., in the culture medium outside the yeast cell. In some embodiments, the cultured broth is centrifuged and the supernatant is removed for assaying the activity of the polypeptide, e.g., the ability to hydrolyze pNPP.

Process for preparing polypeptides having glycerol mono-diacylate lipase activityApplications of

The polypeptides of the present application are polypeptides having lipase activity, in particular glycerol mono-diacylate lipase (MDGL) activity, which are capable of producing glycerol mono-acyls (MAG) of high industrial value by means of esterification or transesterification reactions.

The polypeptides disclosed herein have high enzymatic activity. In some embodiments, the polypeptides disclosed herein are at least about 10% more active, e.g., about 40% more active, than a control polypeptide represented by SEQ ID No. 4. In some embodiments, the polypeptides disclosed herein are at least about 10% more active, e.g., about 30% more active, than commercial enzymes such as Amano G50-K.

The polypeptides disclosed herein may be used for MAG synthesis and hydrolysis of TAG. Accordingly, the present application provides the use of a polypeptide disclosed herein for the preparation of a monoglyceride. In some embodiments, the use of the enzymes disclosed herein can significantly reduce the amount of enzyme added and the cost of production.

In this specification and claims, the words "comprise", "comprising" and "contain" mean "including but not limited to", and are not intended to exclude other moieties, additives, components, or steps.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The above disclosure generally describes the present application, which is further exemplified by the following examples. These examples are described merely to illustrate the present application and do not limit the scope of the present application. Although specific terms and values are employed herein, they are to be understood as exemplary and not limiting the scope of the application.

Examples

Example 1: synthesis and cloning of MDGL sequences

The amino acid sequence of SEQ ID NO:3 (the coding sequence of which is the polypeptide shown in SEQ ID NO: 4) was synthesized by Biotechnology engineering (Shanghai) Inc.

The synthesized MDGL sequence was cloned in plasmid pUC57, stored in E.coli DH 5. Alpha. And the plasmid was digested with AvrII and EcoRI from Fermentas, while plasmid pAO815 carrying constitutive promoter SPI and yeast. Alpha. -mating factor was digested with AvrII and EcoRI, and then purified using the gel recovery kit from Axygen. The MDGL sequence restriction fragment and the plasmid restriction fragment were ligated together using T4DNA ligase from Fermentas according to the product instructions, and the ligation was transformed into E.coli DH 5. Alpha. By heat shock and cultured overnight on LB medium plates containing 100. Mu.g/ml ampicillin. The next day, the single clone was picked up and cultured in LB liquid medium, and plasmids were extracted using the plasmid extraction kit from Axygen and submitted to Shanghai's work for sequencing.

After the recombinant expression vector with correct sequencing is cut and linearized by Bgl II restriction enzyme, a pichia pastoris GS115 competent cell is transformed by an electric shock method, coated on a selection medium MGYS screening plate, and cultured for 3 days at 30 ℃. Transformants were picked and transferred to glycerol mono-diester plates (1% YNB,2% glycerol mono-diacyl ester, 2% agarose, 0.0001% rhodamine B), and cultured at 30 ℃ for 3 days, and the transformant with the largest hydrolysis loop was picked.

Example 2: fermentation verification of glycerol mono-diacyl lipase producing strains

The larger transparent circles of the single clones on the glycerol mono-diacyl ester plate were picked and transferred to a 250ml Erlenmeyer flask containing 50ml of BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) containing no amino acids, ammonium sulfate, 1% glycerol, 4X 10-5% D-biotin, 100mM citric acid-sodium citrate buffer, pH 6.6), and incubated at 30 ℃ for 3 days with shaking at 200 rpm. After the culture, all the bacterial solutions were collected, centrifuged at 8000rpm for 5 minutes, all the supernatants were collected, ultrafiltered using a 10K filter (Millipore), and the protein was concentrated to 20 to 30 times the original fermentation broth, and the protein concentration was measured using Bradford reagent (Biotechnology, shanghai, ltd.) and was 1 to 3mg/ml. The activated strain solution in the flask was inoculated into a 7.5L fermentor, fermented for 136 hours, and the protein concentration and the enzyme activity at different time points were measured, and the results are shown in FIG. 1.

Example 3: detection of specific enzyme activity of MDGL produced by GS115-MDGL by pNPP method

Mu.l of the enzyme solution was added to 400. Mu.l of the pNPP reaction solution (3 mg/mL in isopropanol, 1mL before use and 9mL of a 0.2M sodium acetate-acetic acid solution mixed together), reacted at 35 ℃ for 30min, quenched with 400ul of stop buffer (200 mM Tris-HCL,5% triton-100, pH 8.5), centrifuged at 12000rpm for 5min, and the supernatant was collected and the absorbance (OD) at 405nm was measured.

The enzyme activity unit =0.1935 OD V1 dilution times/T/V2, wherein V1 is the volume (ml) of the reaction solution, V2 is the volume (ml) of the enzyme, T is the reaction time (min), and the dilution times are the dilution times of the enzyme solution. The specific enzyme activity is the ratio of the unit of enzyme activity (U) to the amount of enzyme protein (mg).

The specific enzyme activity of MDGL obtained by triangular flask fermentation was determined to be about 20U/mg.

Example 4: MDGL random PCR mutation and high specific enzyme activity mutant screening

The MDGL portion of the plasmid containing MDGL was amplified using primers EPG50-1 (SEQ ID NO.5: gcgCCTAGGCGGCGAAACGATGATTTCCTTCAATTTT) and EPG50-2 (SEQ ID NO.6: ccgGAATTCTTAAACACACGTTTGAATG), PCR was performed using Taq enzyme from TaKaRa, and the reaction system was: 10 XBuffer 5. Mu.l, dNTP mix (2.5 mM each) 4. Mu.l, primers 1. Mu.l each, plasmid template 0.5. Mu.l, taq enzyme 0.5. Mu.l, mnCl2 added in addition at 0.3mM, double distilled water to 50. Mu.l. The PCR reaction procedure was 98 ℃ for 10s,94 ℃ for 20s,56 ℃ for 20s,72 ℃ for 1min,72 ℃ for 5min, and 30 cycles. The PCR product was purified using the Axygen PCR purification kit. The purified PCR product was digested with Sac-II and EcoRI restriction sites and cloned to the corresponding restriction sites of P0586-G50, and the resulting vector was transformed into E.coli DH 5. Alpha. Strain. Each 1 × 10 ³ Each MDGL mutant was washed with 2ml of sterile water to 8ml of LB liquid medium (containing 100. Mu.g/ml ampicillin), and cultured at 37 ℃ for 4 hours. Plasmid was extracted using the Axygen miniprep plasmid extraction kit, linearized with SalI, and a fragment of about 8.5kb was recovered, transformed into Pichia pastoris GS115 competent cells by electroporation, spread onto MGYS selection plates, and cultured at 30 ℃ for three days. Selecting transformants, transferring the transformants to a glycerol mono-diacylate plate, culturing the transformants at 30 ℃ for 2 days, selecting the transformant with the largest hydrolysis loop, and inoculating the transformant to the culture mediummu.L of BMGY medium was incubated at 30 ℃ for 2 days with shaking at 200 rpm. The cultured cell suspension was centrifuged at 12000rpm for 5min, and the supernatant was aspirated and stored at 4 ℃ for determination of pNPP hydrolysis ability.

MDGL with high specific enzyme activity was screened using 96-well plates. The reaction system of the 96-pore plate is as follows: mu.L of the enzyme solution was added to 200. Mu.L of the pNPP reaction solution (6 mg/mL of the pNPP isopropanol solution, 1mL taken before use and 9mL of the reaction solution (200 mM sodium acetate-acetic acid, pH 4.84) and reacted at 35 ℃ for 30min, 100. Mu.L of a stop solution (200 mM Tris-HCl,5% triton-100, pH 8.5) was added to stop the reaction, and the reaction mixture was centrifuged at 4000rpm for 5min to collect the supernatant and the absorbance at 405nm was measured. Selecting a mutant with the largest specific enzyme activity, and verifying the mutant in a 1.5ml centrifuge tube again, wherein the reaction system comprises the following steps: adding 10 mul of enzyme solution into 400 mul of pNPP reaction solution, reacting for 30min at 35 ℃, adding 400 mul of stop solution, centrifuging for 5min at 4000rpm, taking supernatant, measuring absorbance at 405nm, and calculating specific enzyme activity. The method for calculating the specific enzyme activity was the same as that in example 3.

Example 5: high specific enzyme activity mutant triangular flask fermentation verification

In this example, the mutants obtained from the preliminary screening in example 4 were subjected to validation by flask fermentation. The specific operation steps are as follows: the selected mutants were inoculated into 50mL of BMGY medium, incubated at 30 ℃ for 2 days with shaking at 200rpm, and supplemented with 0.5mL of glycerol every 12h in 50mL of the medium. After 3 days of culture, the fermentation broth was centrifuged at 8000rpm at 4 ℃, the supernatant was filtered through a 0.22 μm filter, an equal amount of the supernatant was concentrated to the same volume using a Milipore 10Kda ultrafiltration concentration tank, the protein concentration was measured using Bradford's reagent, the specific activity of MDGL in each mutant fermentation broth was measured using the pNPP method, mutants having the highest specific activity were selected, and the enzyme solutions having equal protein amounts and equal enzyme activity units were subjected to polyacrylamide gel electrophoresis analysis, and the results are shown in FIG. 2.

Example 6: high specific enzyme activity mutant sequence determination

In this example, the sequence of the mutant with high specific enzyme activity, MDGL-new, screened and verified in examples 4 and 5 was determined.

The sequence of MDGL-new was amplified using primers SPI-F (SEQ ID NO.7: GGCGAAACGATGAGATTTCCT) and PLC-R (SEQ ID NO.8: GTGCCGAGGATGACGATGAG), PCR was performed using Taq enzyme from TaKaRa, and the PCR product was sequenced by Biotech Ltd. The nucleotide sequence of MDGL-new is shown as SEQ ID NO:2, the amino acid sequence of MDGL-new is shown as SEQ ID NO:1 is shown.

Example 7: purification and validation of MDGL-new encoding Gene

The MDGL-new nucleotide sequence sequenced in example 6 was amplified using primers PLC-FA (SEQ ID NO.9: CCCTAGGTGGTCAGCTGAGGACAAGCATAA) and PLC-RB (SEQ ID NO.10: CGGGATCCGTGCGAGGATGACGATGAG), cleaved with Sac-II and EcoRI and cloned into P0586-G50, the resulting vector was transformed into E.coli DH 5. Alpha. Strain, and a single clone was picked for sequencing verification and considered as a positive clone if it was completely identical to the MDGL-new nucleotide sequence. The positive clone was extracted into a plasmid, linearized with SalI, recovered as a fragment of about 8.5kb, and transformed into Pichia pastoris GS 115. Transformants were picked on a glycerol mono-diacylate plate, cultured at 30 ℃ for 3 days, and the transformant with the largest hydrolysis loop was picked for triangular flask fermentation. Then, the specific enzyme activity of MDGL in the fermentation broth was measured by the pNPP method using the same method as in example 5, wherein the measured specific enzyme activity was 58.4U/mg.

Example 8: MDGL-new activity unit determination

In this example, the enzyme activity units of the commercial enzyme Amano G50-K (available from Tianye enzyme preparation (Jiangsu) Co., ltd.), the polypeptide shown in SEQ ID NO:4 and MDGL-new were determined using vinyl laurate as a substrate, and the reaction system was: 500 μ L of substrate (3% vinyl laurate, 2% PVA), 400 μ L of 0.1M sodium acetate (pH 5.6), 100 μ L of diluted enzyme solution (6 mg/mL protein, diluted 20000 times), resting reaction at 37 ℃ for 30min, and addition of 4mL of stop solution (ethanol: acetone = 1. Titrating the reaction solution by using 0.01M ethanol potassium hydroxide, taking phenolphthalein as an indicator,

the unit of enzyme activity is defined as: the amount of protein required to release 1 μ M lauric acid per minute was 1U. Calculating the formula: the unit of enzyme activity = (V1-V2) × 0.01 × 106 × d/30, V1: amount of potassium hydroxide consumed by the sample, V2: amount of potassium hydroxide consumed for blank, D: and (4) dilution times.

The measurement results are as follows: the specific enzyme activity of Amano G50-K is 6256.41U/mg; the specific enzyme activity of the reference MDGL shown as SEQ ID NO. 4 is 5687.83U/mg, which is 90.9% of Amano commercial enzyme; the specific enzyme activity of MDGL-new is 8045.98U/mg, which is 28.6% higher than that of commercial enzyme and 41.5% higher than that of control MDGL.

It is to be understood that while the application is illustrated in certain forms, it is not limited to what has been shown and described herein. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the application. Such variations are within the scope of the claims of this application.

Sequence listing

<110> Fengyi (Shanghai) Biotechnology research and development center, ltd

<120> novel glycerol mono-diacyl ester lipase

<130> 17C13550CN

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<170> SIPOSequenceListing 1.0

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Asp Val Ser Thr Ser Glu Leu Asp Gln Phe Glu Phe Trp Val Gln Tyr

1 5 10 15

Ala Ala Ala Ser Tyr Tyr Glu Ala Asp Tyr Thr Ala Gln Val Gly Asp

20 25 30

Lys Leu Ser Cys Ser Lys Gly Asn Cys Pro Glu Val Glu Ala Thr Gly

35 40 45

Ala Thr Val Ser Tyr Asp Phe Ser Asp Ser Thr Ile Thr Asp Thr Ala

50 55 60

Gly Tyr Ile Ala Val Asp His Thr Asn Ser Ala Val Val Leu Ala Phe

65 70 75 80

Arg Gly Ser His Ser Val Arg Asn Trp Val Ala Asp Ala Thr Phe Val

85 90 95

His Thr Asn Pro Gly Leu Cys Asp Gly Cys Leu Ala Glu Leu Gly Phe

100 105 110

Trp Ser Ser Trp Lys Leu Val Arg Asp Asp Ile Ile Lys Glu Leu Lys

115 120 125

Glu Val Val Ala Gln Asn Pro Asp Tyr Glu Leu Val Val Val Gly His

130 135 140

Ser Leu Gly Ala Ala Val Ala Thr Leu Ala Ala Thr Asp Leu Arg Gly

145 150 155 160

Lys Gly Tyr Pro Ser Ala Lys Leu Tyr Ala Tyr Ala Ser Pro Arg Val

165 170 175

Gly Asn Ala Ala Leu Ala Lys Tyr Ile Thr Ala Gln Gly Asn Asn Phe

180 185 190

Arg Phe Thr His Thr Asn Asp Pro Val Pro Lys Leu Pro Leu Leu Ser

195 200 205

Met Gly Tyr Val His Val Ser Pro Glu Tyr Trp Ile Thr Ser Pro Asn

210 215 220

Asn Ala Thr Val Ser Thr Ser Asp Ile Lys Val Ile Asp Gly Asp Val

225 230 235 240

Ser Phe Asp Gly Asn Thr Gly Thr Gly Leu Pro Leu Leu Thr Asp Phe

245 250 255

Glu Ala His Ile Trp Tyr Phe Val Gln Val Asp Ala Gly Lys Gly Pro

260 265 270

Gly Leu Pro Phe Lys Arg Val

275

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<213> Artificial Sequence (Artificial Sequence)

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tgcccagaag ttgaagcaac cggtgcaact gtgtcttacg acttctccga ttccacgatc 180

actgacaccg caggttacat cgcagttgat cacaccaact ccgcagtggt actggcattc 240

cgtggttctc actccgtacg taactgggtt gctgatgcta ctttcgtcca taccaaccca 300

ggtctgtgtg atggttgcct ggctgagctg ggtttctggt cttcctggaa gctggttcgt 360

gatgatatta tcaaagaact gaaagaagtg gtggcacaga acccagacta tgaactggtg 420

gtcgtgggcc actccctggg tgctgctgtg gctactctgg ctgctaccga cctgcgtggt 480

aaaggttatc catctgctaa actgtacgcc tacgcttccc ctcgtgttgg caacgcagcc 540

ctggccaaat atatcaccgc ccagggcaac aacttccgtt tcacccacac caatgaccca 600

gtacctaaac tgccactgct gtctatgggc tatgtacatg tttctcctga atattggatc 660

acctctccta acaacgccac tgtttctacc tctgacatca aagtcattga cggcgacgta 720

tcttttgacg gcaataccgg cacgggcctg cctctgctga cggactttga agcccacatt 780

tggtactttg tacaggttga cgccggcaaa ggtcctggcc tgccattcaa acgtgtttaa 840

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<213> Artificial Sequence (Artificial Sequence)

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gatgtctcca cttccgaact ggaccagttc gagttctggg ttcaatacgc agccgcctct 60

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actgacaccg caggttacat cgcagttgat cacaccaact ccgcagtggt actggcattc 240

cgtggttctt actccgtacg taactgggtt gctgatgcta ctttcgtcca taccaaccca 300

ggtctgtgtg atggttgtct ggctgagctg ggtttctggt cttcctggaa gctggttcgt 360

gatgatatta tcaaagaact gaaagaagtg gtggcacaga acccaaacta tgaactggtg 420

gtcgtgggcc actccctggg tgctgctgtg gctactctgg ctgctaccga cctgcgtggt 480

aaaggttatc catctgctaa actgtacgct tacgcttccc ctcgtgttgg caacgcagcc 540

ctggccaaat atatcaccgc ccagggcaac aacttccgtt tcacccacac caatgaccca 600

gtacctaaac tgccactgct gtctatgggc tatgtacatg tttctcctga atattggatc 660

acctctccta acaacgccac tgtttctacc tctgacatca aagtcattga cggcgacgta 720

tcttttgacg gcaataccgg cacgggcctg cctctgctga cggactttga agcccacatt 780

tggtactttg tacaggttga cgccggcaaa ggtcctggcc tgccattcaa acgtgtttaa 840

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<211> 279

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<213> Artificial Sequence (Artificial Sequence)

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Asp Val Ser Thr Ser Glu Leu Asp Gln Phe Glu Phe Trp Val Gln Tyr

1 5 10 15

Ala Ala Ala Ser Tyr Tyr Glu Ala Asp Tyr Thr Ala Gln Val Gly Asp

20 25 30

Lys Leu Ser Cys Ser Lys Gly Asn Cys Pro Glu Val Glu Ala Thr Gly

35 40 45

Ala Thr Val Ser Tyr Asp Phe Ser Asp Ser Thr Ile Thr Asp Thr Ala

50 55 60

Gly Tyr Ile Ala Val Asp His Thr Asn Ser Ala Val Val Leu Ala Phe

65 70 75 80

Arg Gly Ser Tyr Ser Val Arg Asn Trp Val Ala Asp Ala Thr Phe Val

85 90 95

His Thr Asn Pro Gly Leu Cys Asp Gly Cys Leu Ala Glu Leu Gly Phe

100 105 110

Trp Ser Ser Trp Lys Leu Val Arg Asp Asp Ile Ile Lys Glu Leu Lys

115 120 125

Glu Val Val Ala Gln Asn Pro Asn Tyr Glu Leu Val Val Val Gly His

130 135 140

Ser Leu Gly Ala Ala Val Ala Thr Leu Ala Ala Thr Asp Leu Arg Gly

145 150 155 160

Lys Gly Tyr Pro Ser Ala Lys Leu Tyr Ala Tyr Ala Ser Pro Arg Val

165 170 175

Gly Asn Ala Ala Leu Ala Lys Tyr Ile Thr Ala Gln Gly Asn Asn Phe

180 185 190

Arg Phe Thr His Thr Asn Asp Pro Val Pro Lys Leu Pro Leu Leu Ser

195 200 205

Met Gly Tyr Val His Val Ser Pro Glu Tyr Trp Ile Thr Ser Pro Asn

210 215 220

Asn Ala Thr Val Ser Thr Ser Asp Ile Lys Val Ile Asp Gly Asp Val

225 230 235 240

Ser Phe Asp Gly Asn Thr Gly Thr Gly Leu Pro Leu Leu Thr Asp Phe

245 250 255

Glu Ala His Ile Trp Tyr Phe Val Gln Val Asp Ala Gly Lys Gly Pro

260 265 270

Gly Leu Pro Phe Lys Arg Val

275

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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ccggaattct taaacacgtt tgaatg 26

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<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggcgaaacga tgagatttcc t 21

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<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtgccgagga tgacgatgag 20

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccctaggtgg tcagctgagg acaagcataa 30

<210> 10

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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Claims (11)

1. A polypeptide having glycerol mono-diacyl ester lipase activity, which consists of an amino acid sequence as shown in SEQ ID NO 1.

2. The polypeptide of claim 1, which is capable of being fused to a heterologous polypeptide, wherein the heterologous polypeptide is selected from a purification tag, an epitope tag, a targeting sequence, or a signal peptide.

3. A polynucleotide encoding the polypeptide of claim 1.

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

5. An expression vector comprising the polynucleotide of claim 3 or 4.

6. The expression vector of claim 5, further comprising a control sequence that regulates expression of the polynucleotide, wherein the polynucleotide is operably linked to the control sequence.

7. The expression vector of claim 5 or 6, which is a plasmid.

8. A host cell comprising the polynucleotide of claim 3 or 4 or the expression vector of any one of claims 5-7.

9. The host cell of claim 8, which is a yeast or E.

10. Use of the polypeptide of claim 1 or 2 for the preparation of monoglycerides.

11. A method of synthesizing a monoacylglycerol, comprising contacting the polypeptide of claim 1 or 2 with a fatty acid and glycerol.

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