CN113005111B - Novel glycerol mono-diacyl ester lipase - Google Patents

Novel glycerol mono-diacyl ester lipase Download PDF

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CN113005111B
CN113005111B CN202011499617.4A CN202011499617A CN113005111B CN 113005111 B CN113005111 B CN 113005111B CN 202011499617 A CN202011499617 A CN 202011499617A CN 113005111 B CN113005111 B CN 113005111B
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赵强
徐正军
牛其文
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Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
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Abstract

The present application provides novel polypeptides having monoglyceride lipase activity comprising an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 3 and 15 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 containing the polynucleotides. Furthermore, the present application relates to the use of the above-described polypeptide having monoglyceride-diacyl lipase activity.

Description

Novel glycerol mono-diacyl ester lipase
Cross Reference to Related Applications
The present application claims priority from chinese patent application No. 201911329804.5 filed on date 20 and 12 in 2019, which is incorporated herein by reference in its entirety.
Technical Field
The present application is in the field of enzyme engineering, in particular, polypeptides having lipase activity, nucleic acids encoding the same, and expression vectors and host cells comprising the same. The application also relates to a screening method and application of the polypeptide.
Background
Lipase is one of esterases and can catalyze the hydrolysis of ester bonds of triglycerides, diglycerides, monoglycerides, other small molecule esters, polyol esters and polybasic acid esters, fat is a natural substrate of lipase, diglycerides and monoglycerides are produced in the hydrolysis process, and hydrolysis end products are glycerol and fatty acids. The lipase has mild hydrolysis condition, less byproducts and no coenzyme. Fat is mostly a hydrophobic substance, so that hydrolysis occurs at the oil-water interface or in the organic phase. The monoglyceride-diacylglycerol lipase (MDGL) is one of lipases, the MDGL has substrate specificity, only acts on Monoglyceride (MAG) and Diglyceride (DAG), has no catalysis effect on triacylglycerol, and can produce monoglyceride with high industrial value by esterification or transesterification reaction.
MAG is an excellent emulsifier and has wide application in the food, pharmaceutical and cosmetic industries. The traditional synthesis process of MAG is to produce through continuous esterification of grease/Triglyceride (TAG) and glycerin under the condition of nitrogen at high temperature by taking inorganic base as a catalyst, wherein the product is a mixture of MAG, DAG and TAG, and the MAG is obtained through distillation, and the yield of the MAG is 40-50%. Unlike traditional synthesis process, the enzymatic synthesis of MAG takes fatty acid and glycerin as substrates, and the catalytic synthesis of MAG is carried out under mild condition, wherein the conversion rate of fatty acid in enzymatic catalysis is more than 97%, and the MAG accounts for more than 74% of the product. The main problems of the current restriction enzyme method application are lower enzyme activity and higher enzyme cost.
Summary of The Invention
In a first aspect, there is provided a polypeptide having glycerol mono-diacyl ester lipase activity comprising or consisting of a sequence selected from the group consisting of seq id nos:
(a) An amino acid sequence as shown in any one of SEQ ID NO 1-3 and 15; and
(b) A sequence obtained by substituting, deleting or adding at least one amino acid into the sequence of (a); wherein the polypeptide variant obtained from (b) retains monoglyceride-diacyl lipase activity.
In some embodiments, the polypeptides of the present application comprise an amino acid sequence set forth in any one of SEQ ID NOs 1-3 and 15. In a preferred embodiment, the above polypeptide consists of the amino acid sequence shown in any one of SEQ ID NOs 1 to 3 and 15. In a more preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO. 2, 3 or 15.
Herein, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1 is designated MDGLs, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 2 is designated MDGLc, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 3 is designated MDGLsc, and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 15 is designated MDGL-3G3.
In a second aspect, there is provided a polynucleotide encoding the polypeptide of the first aspect comprising or consisting of a sequence selected from the group consisting of seq id nos:
(a) A nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NOs 1 to 3 and 15 or a sequence comprising at least one amino acid substitution, deletion or addition in the above sequence; and
(b) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of (a).
In some embodiments, the polynucleotides of the present application comprise a nucleotide sequence set forth in any one of SEQ ID NOs 4-6 and 16. In a preferred embodiment, the above polynucleotide consists of the nucleotide sequence shown in any one of SEQ ID NOS.4-6 and 16. In a more preferred embodiment, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO. 5, 6 or 16.
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 regulatory sequence that regulates the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence. In a preferred embodiment, the expression vector is a plasmid vector, such as pPIC9K.
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 pastoris, or e.
In a fifth aspect, there is provided the use of a polypeptide according to the first aspect for catalyzing a synthesis, hydrolysis or transesterification reaction of an ester.
In some embodiments, the esters are monoglycerides and/or diglycerides.
In a sixth aspect, there is provided a method of synthesizing a monoglyceride and/or diglyceride 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 a polypeptide of the present application comprising:
1) Mutating the nucleotide sequence shown in SEQ ID NO. 7 to obtain a mutant sequence;
2) Expressing the mutated sequence obtained in step 1) to obtain a polypeptide mutated with respect to the polypeptide shown in SEQ ID NO. 8; and
3) Mutant polypeptides with enzyme activity and/or thermal stability higher than those of the polypeptide shown in SEQ ID NO. 8 are screened.
In addition, the application also provides the use of the nucleotide sequence shown in SEQ ID NO. 7 for screening the polypeptide disclosed herein.
The polypeptides obtained by the screening methods disclosed herein have higher lipase activity (in particular glycerol mono-diacyl ester lipase activity) and better thermostability.
Brief description of the drawings
FIG. 1 shows the results of MDGL mutant delta flask fermentation protein production.
FIG. 2 shows the results of the enzymatic yield of MDGL mutant delta flask fermented protein.
FIG. 3 shows the results of the thermostability of MDGL mutants.
FIG. 4 shows the result of SDS-PAGE analysis of MDGL mutants.
In the above figures, s represents MDGLs, c represents MDGLc, and sc represents MDGLsc.
FIG. 5 shows the results of protein production by MDGL-3G3 mutant delta flask fermentation.
FIG. 6 shows the results of the enzyme activity yield and specific enzyme activity of MDGL-3G3 mutant delta flask fermented protein.
FIG. 7 shows the results of thermostability of MDGL-3G3 mutants.
FIG. 8 shows the result of SDS-PAGE analysis of MDGL-3G3 mutant.
Brief description of the sequence
SEQ ID NO. 1: amino acid sequence of MDGLs;
SEQ ID NO. 2: amino acid sequence of MDGLc;
SEQ ID NO. 3: the amino acid sequence of MDGLsc;
SEQ ID NO. 4: coding nucleic acid sequences for MDGLs;
SEQ ID NO. 5: a nucleic acid sequence encoding MDGLc;
SEQ ID NO. 6: a nucleic acid sequence encoding MDGLsc;
SEQ ID NO. 7: an optimized MDGL coding nucleic acid sequence;
SEQ ID NO. 8: an optimized MDGL amino acid sequence;
SEQ ID NO. 9: forward amplification primer MDGLm-F1;
SEQ ID NO. 10: reverse amplification primer MDGLm-R1;
SEQ ID NO. 11: forward amplification primer MDGLm-F2;
SEQ ID NO. 12: reverse amplification primer MDGLm-R2;
SEQ ID NO. 13: forward amplification primer MDGL-ctlm-F;
SEQ ID NO. 14: reverse amplification primer MDGL-ctlm-R;
SEQ ID NO. 15: an amino acid sequence of MDGL-3G 3;
SEQ ID NO. 16: a nucleic acid sequence encoding MDGL-3G 3.
Detailed Description
Polypeptides of the present application
The present application provides polypeptides having lipase activity, in particular glycerol mono-diacyl ester lipase activity, comprising or consisting of a sequence selected from the group consisting of seq id nos:
(a) An amino acid sequence as shown in any one of SEQ ID NO 1-3 and 15; and
(b) A sequence obtained by substituting, deleting or adding at least one amino acid into the sequence of (a); wherein the polypeptide variant obtained from (b) retains lipase activity.
Glycerol mono-diacyl ester lipase (MDGL) is one type of lipase. MDGL has 2 forms, the molecular weight is 37kDa and 39kDa respectively, MDGL has a signal peptide consisting of 26 amino acids, and the mature peptide contains 279 amino acids. For example, MDGL enzyme activity units can be determined using vinyl laurate as a substrate.
In certain embodiments, the number of amino acid substitutions, deletions or additions described above 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 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 variants differ from the amino acid sequence shown in SEQ ID NO. 1 by about 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.
In preferred embodiments, the above polypeptide variants differ 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 variants differ 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.
In preferred embodiments, the above polypeptide variants differ from the amino acid sequence shown in SEQ ID NO. 3 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 variants differ from the amino acid sequence shown in SEQ ID NO. 3 by about 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.
In preferred embodiments, the above polypeptide variants differ from the amino acid sequence shown in SEQ ID NO. 15 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 variants differ from the amino acid sequence shown in SEQ ID NO. 15 by about 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.
In some embodiments, the polypeptides of the present application comprise an amino acid sequence set forth in any one of SEQ ID NOs 1-3 and 15. In a preferred embodiment, the above polypeptide consists of the amino acid sequence shown in any one of SEQ ID NOs 1 to 3 and 15. In a more preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO. 2, 3 or 15.
Herein, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1 is designated MDGLs, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 2 is designated MDGLc, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 3 is designated MDGLsc, and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 15 is designated MDGL-3G3.
The amino acid sequence of SEQ ID NO. 1 of the present application is shown below:
DVSTSELDQFEFWVQYAAASYCEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFSDSTITDTAGYIAVDHTNSAVVLAFRGSYSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAYASPRVGNAALAKYITAQGNNFRFTHTNDPVPKLPLLSMGYVHVSPEYWITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDACKGPGLPFKRV
the amino acid sequence of SEQ ID NO. 2 of the present application is shown below:
DVSTSELDQFEFWVQYAAASYYEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFSDSTITDTAGYIAVDHTNSAVVLAFRGSYSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAYASPRVGNAALAKYITAQGNNFRFTHTNDPVPKLPLLSMGYVHVSPEYWITSPNNATVRRRDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAGKGPGLPFKRV
the amino acid sequence of SEQ ID NO. 3 of the present application is shown below:
DVSTSELDQFEFWVQYAAASYCEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFSDSTITDTAGYIAVDHTNSAVVLAFRGSYSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAYASPRVGNAALAKYITAQGNNFRFTHTNDPVPKLPLLSMGYVHVSPEYWITSPNNATVRRRDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDACKGPGLPFKRV
the amino acid sequence of SEQ ID NO. 15 of the present application is shown below:
DVSTSELDQFEFWVQYAAASYCEADYRAQVGDKLSCSKGNCPEVEATGATVSYDFSDSTITDTAGYIAVDHTNSAVVLAFRGSHSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKELKEVVAQNPDYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAYASPRVGNAALAKYITAQGNNFRFTHTNDPVPKLPLLSMGYVHVSPEYWITSPNNATVRRRDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDACKGPGLPFKRV
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, desmin, isodesmin, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D) -amino acids, norleucine, norvaline, p-fluorophenylalanine, ethylsulfanilic acid, 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 acid, or derivatization of the amino acid. Amino acid mimics include, for example, organic structures that exhibit functionally similar properties, such as charge and charge-space characteristics of amino acids. For example, the 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 a naturally occurring Arg amino acid. The mimetic also includes a constraining structure to maintain optimal space and charge interactions of the 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 mimics.
In some embodiments, the variant of the amino acid sequence shown in SEQ ID NO. 1 has 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.
In some embodiments, the variant of the amino acid sequence shown in SEQ ID NO. 2 has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with SEQ ID NO. 2. In a preferred embodiment, the polypeptide variant has more than 99% homology with the sequence shown in SEQ ID NO. 2.
In some embodiments, the variant of the amino acid sequence shown in SEQ ID NO. 3 has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with SEQ ID NO. 3. In a preferred embodiment, the polypeptide variant has more than 99% homology with the sequence shown in SEQ ID NO. 3.
In some embodiments, the variant of the amino acid sequence shown in SEQ ID NO. 15 has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with SEQ ID NO. 15. In a preferred embodiment, the polypeptide variant has more than 99% homology with the sequence shown in SEQ ID NO. 15.
"homology" as used 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 desired, to achieve a maximum percentage of homology. Methods and computer programs for alignment are well known in the art.
"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 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 the polypeptide fragments shown in any of SEQ ID NO 1-3 and 15.
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 any of SEQ ID NO's 1-3 and 15, wherein the substituted sequence still retains similar lipase catalytic activity, in particular glycerol mono-diacyl ester lipase activity.
Certain amino acid substitutions, known as "conservative amino acid substitutions," can frequently occur in a protein without altering the conformation or function of the protein, which is an established rule in protein chemistry.
Conservative amino acid substitutions in this application include, but are not limited to, substitution of any one of glycine (G), alanine (a), isoleucine (I), valine (V), and leucine (L) for any other one of these aliphatic amino acids; substitution of serine (S) for threonine (T) 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 other of these aromatic amino acids with phenylalanine (F), tyrosine (Y) and tryptophan (W); and substitution of cysteine (C) with methionine (M) 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 with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchanged at the following positions: wherein the important feature of the amino acid residues is their charge and the different pK of the two amino acid residues is not apparent. Under certain circumstances, there are still other variations that may be considered "conservative" (see, e.g., BIOCHEMISTRIY 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)。
Hereinafter, amino acid residues are exemplified by a classification 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, t-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;
group F: serine, threonine and homoserine;
group G: phenylalanine and tyrosine.
In other specific embodiments, the C-terminal or N-terminal region of a polypeptide of the present application, e.g., a polypeptide set forth in any one of SEQ ID NOs 1-3 and 15, may also be truncated by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar lipase catalytic activity, particularly monoglyceride-diacyl lipase activity.
In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added to the C-terminal or N-terminal regions of a polypeptide of the present application, e.g., a polypeptide represented by any one of SEQ ID NOs 1-3 and 15, resulting in a polypeptide variant that still retains similar lipase catalytic activity, particularly monoglyceride-diacyl lipase activity.
Furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in a region other than the C-terminus or N-terminus of a polypeptide, such as any of the sequences set forth in SEQ ID NOs 1-3 and 15, provided that the altered polypeptide substantially retains similar lipase catalytic activity, particularly monoglyceride-diacyl lipase activity.
In certain embodiments, a polypeptide of the present application, such as a polypeptide set forth in any one of SEQ ID NOS: 1-3 and 15, or a variant thereof, is fused to a heterologous polypeptide. In some embodiments, the fusion protein substantially retains the lipase activity of the polypeptide shown in any one of SEQ ID NOs 1-3 and 15. In certain embodiments, the heterologous polypeptide is linked to the N-terminus of the polypeptide shown in any one of SEQ ID NOs 1-3 and 15. In certain embodiments, the heterologous polypeptide is linked to the C-terminus of the polypeptide shown in any one of SEQ ID NOs 1-3 and 15. In these embodiments, the heterologous polypeptide may be selected from a purification tag (which may include, for example, but not limited to, GST, MBP), an epitope tag (which may include, for example, but not limited to, myc, FLAG), a targeting sequence, a signal peptide, and the like. In a specific embodiment, the fusion protein comprises a polypeptide as set forth in any one of SEQ ID NO 1-3 and 15 and a tag, typically a peptide tag, that binds to the C-terminus or N-terminus of the polypeptide as set forth in any one of SEQ ID NO 1-3 and 15. 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 polypeptides disclosed herein comprising or consisting of a sequence selected from the group consisting of seq id nos: (a) A nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NOs 1 to 3 and 15 or a sequence comprising at least one amino acid substitution, deletion or addition in the above sequence; and
(b) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of (a).
In certain specific embodiments, the polynucleotides of the present application encode polypeptides represented by any one of SEQ ID NOs 1-3 and 15, as well as functionally equivalent variants thereof. In some embodiments, 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 polypeptides set forth in any of SEQ ID NOs 1-3 and 15, and functionally equivalent variants thereof.
In certain embodiments, polynucleotides of the present application comprise nucleotide sequences having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the nucleotide sequences set forth in any one of SEQ ID NOs 4-6 and 16. In a preferred embodiment, the polynucleotides of the present application comprise a nucleotide sequence set forth in any one of SEQ ID NOs 4-6 and 16.
The nucleotide sequence of SEQ ID NO. 4 is shown below:
GATGTCTCCACTTCCGAACTGGACCAGTTCGAGTTCTGGGTTCAATACGCAGCCGCCTCTTACTGTGAGGCTGATTACACCGCACAGGTTGGTGATAAGCTGTCCTGCTCTAAGGGTAACTGCCCAGAAGTTGAAGCAACCGGTGCAACTGTGTCTTACGACTTCTCCGATTCCACGATCACTGACACCGCAGGTTACATCGCAGTTGATCACACCAACTCCGCAGTGGTACTGGCATTCCGTGGTTCTTACTCCGTACGTAACTGGGTTGCTGATGCTACTTTCGTCCATACCAACCCAGGTCTGTGTGATGGTTGTCTGGCTGAGCTGGGTTTCTGGTCTTCCTGGAAGCTGGTTCGTGATGATATTATCAAAGAACTGAAAGAAGTGGTGGCACAGAACCCAAACTATGAACTGGTGGTCGTGGGCCACTCCCTGGGTGCTGCTGTGGCTACTCTGGCTGCTACCGACCTGCGTGGTAAAGGTTATCCATCTGCTAAACTGTACGCTTACGCTTCCCCTCGTGTTGGCAACGCAGCCCTGGCCAAATATATCACCGCCCAGGGCAACAACTTCCGTTTCACCCACACCAATGACCCAGTACCTAAACTGCCACTGCTGTCTATGGGCTATGTACATGTTTCTCCTGAATATTGGATCACCTCTCCTAACAACGCCACTGTTTCTACCTCTGACATCAAAGTCATTGACGGCGACGTATCTTTTGACGGCAATACCGGCACGGGCCTGCCTCTGCTGACGGACTTTGAAGCCCACATTTGGTACTTTGTACAGGTTGACGCCTGCAAAGGTCCTGGCCTGCCATTCAAACGTGTTTAA
the nucleotide sequence of SEQ ID NO. 5 is shown below:
GATGTCTCCACTTCCGAACTGGACCAGTTCGAGTTCTGGGTTCAATACGCAGCCGCCTCTTACTACGAGGCTGATTACACCGCACAGGTTGGTGATAAGCTGTCCTGCTCTAAGGGTAACTGCCCAGAAGTTGAAGCAACCGGTGCAACTGTGTCTTACGACTTCTCCGATTCCACGATCACTGACACCGCAGGTTACATCGCAGTTGATCACACCAACTCCGCAGTGGTACTGGCATTCCGTGGTTCTTACTCCGTACGTAACTGGGTTGCTGATGCTACTTTCGTCCATACCAACCCAGGTCTGTGTGATGGTTGTCTGGCTGAGCTGGGTTTCTGGTCTTCCTGGAAGCTGGTTCGTGATGATATTATCAAAGAACTGAAAGAAGTGGTGGCACAGAACCCAAACTATGAACTGGTGGTCGTGGGCCACTCCCTGGGTGCTGCTGTGGCTACTCTGGCTGCTACCGACCTGCGTGGTAAAGGTTATCCATCTGCTAAACTGTACGCTTACGCTTCCCCTCGTGTTGGCAACGCAGCCCTGGCCAAATATATCACCGCCCAGGGCAACAACTTCCGTTTCACCCACACCAATGACCCAGTACCTAAACTGCCACTGCTGTCTATGGGCTATGTACATGTTTCTCCTGAATATTGGATCACCTCTCCTAACAACGCCACTGTTCGTAGACGTGACATCAAAGTCATTGACGGCGACGTATCTTTTGACGGCAATACCGGCACGGGCCTGCCTCTGCTGACGGACTTTGAAGCCCACATTTGGTACTTTGTACAGGTTGACGCCGGCAAAGGTCCTGGCCTGCCATTCAAACGTGTTTAA
the nucleotide sequence of SEQ ID NO. 6 is shown below:
GATGTCTCCACTTCCGAACTGGACCAGTTCGAGTTCTGGGTTCAATACGCAGCCGCCTCTTACTGTGAGGCTGATTACACCGCACAGGTTGGTGATAAGCTGTCCTGCTCTAAGGGTAACTGCCCAGAAGTTGAAGCAACCGGTGCAACTGTGTCTTACGACTTCTCCGATTCCACGATCACTGACACCGCAGGTTACATCGCAGTTGATCACACCAACTCCGCAGTGGTACTGGCATTCCGTGGTTCTTACTCCGTACGTAACTGGGTTGCTGATGCTACTTTCGTCCATACCAACCCAGGTCTGTGTGATGGTTGTCTGGCTGAGCTGGGTTTCTGGTCTTCCTGGAAGCTGGTTCGTGATGATATTATCAAAGAACTGAAAGAAGTGGTGGCACAGAACCCAAACTATGAACTGGTGGTCGTGGGCCACTCCCTGGGTGCTGCTGTGGCTACTCTGGCTGCTACCGACCTGCGTGGTAAAGGTTATCCATCTGCTAAACTGTACGCTTACGCTTCCCCTCGTGTTGGCAACGCAGCCCTGGCCAAATATATCACCGCCCAGGGCAACAACTTCCGTTTCACCCACACCAATGACCCAGTACCTAAACTGCCACTGCTGTCTATGGGCTATGTACATGTTTCTCCTGAATATTGGATCACCTCTCCTAACAACGCCACTGTTCGTAGACGTGACATCAAAGTCATTGACGGCGACGTATCTTTTGACGGCAATACCGGCACGGGCCTGCCTCTGCTGACGGACTTTGAAGCCCACATTTGGTACTTTGTACAGGTTGACGCCTGCAAAGGTCCTGGCCTGCCATTCAAACGTGTTTAA
the nucleotide sequence of SEQ ID NO. 16 is shown below:
GACGTTTCTACTTCCGAGTTGGACCAATTCGAGTTCTGGGTTCAATACGCTGCTGCTTCTTACTGTGAGGCTGATTACAGAGCTCAAGTTGGAGATAAGTTGTCTTGTTCTAAGGGTAACTGTCCTGAAGTTGAGGCTACTGGTGCTACTGTTTCTTACGATTTCTCCGATTCTACTATCACTGATACTGCCGGTTACATTGCTGTTGATCACACTAACTCTGCTGTTGTTTTGGCTTTCAGAGGTTCTCACTCTGTTCGTAACTGGGTTGCTGACGCTACTTTTGTTCACACTAACCCAGGTTTGTGTGATGGTTGTTTGGCTGAGTTGGGTTTCTGGTCCTCCTGGAAGTTGGTTAGAGATGACATTATTAAGGAGTTGAAGGAGGTTGTCGCTCAAAACCCTGACTACGAGTTGGTTGTTGTTGGTCACTCTTTGGGTGCTGCCGTTGCTACCTTGGCTGCCACTGACTTGAGAGGTAAAGGTTACCCATCTGCTAAGTTGTACGCTTACGCTTCTCCAAGAGTTGGTAACGCTGCTTTGGCTAAGTACATTACCGCTCAAGGTAACAACTTCAGATTCACTCACACTAACGATCCAGTTCCAAAGTTGCCATTGTTGTCCATGGGTTACGTTCACGTTTCTCCAGAGTACTGGATTACTTCCCCAAACAACGCTACTGTTCGTAGACGTGACATTAAGGTTATCGATGGAGATGTTTCTTTCGACGGTAACACTGGTACCGGTTTGCCATTGTTGACTGACTTCGAGGCTCACATCTGGTACTTCGTTCAAGTTGATGCTTGTAAGGGTCCAGGTTTGCCATTCAAGAGAGTTTAA
in a preferred embodiment, 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 sequences shown in any of SEQ ID NO. 4-6 and 16. In a more preferred embodiment, the polynucleotides of the present application consist of the nucleotide sequence shown in any one of SEQ ID NOs 4 to 6 and 16. In a most preferred embodiment, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO. 5, 6 or 16.
In a specific embodiment, the polynucleotide shown in SEQ ID NO. 4 encodes the amino acid sequence shown in SEQ ID NO. 1; the polynucleotide shown in SEQ ID NO. 5 codes for an amino acid sequence shown in SEQ ID NO. 2; the polynucleotide shown in SEQ ID NO. 6 encodes an amino acid sequence shown in SEQ ID NO. 3; the polynucleotide shown in SEQ ID NO. 16 encodes the amino acid sequence shown in SEQ ID NO. 15.
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 nucleotide sequences that specifically hybridize to nucleotide sequences encoding polypeptides set forth in any of SEQ ID NO:1-3 and 15 and functionally equivalent variants thereof under stringent conditions.
Stringent conditions for DNA hybridization can be routinely selected by those skilled in the art. Longer probes typically require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the re-annealing ability of denatured DNA when the complementary strand is in an environment below its melting temperature. The higher the degree of homology between the probe and the 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 severity is lower. For a detailed description of the stringent conditions of 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 dodecyl sulfate at 50deg.C; 2) For hybridization, denaturing agents such as formamide (e.g., 50% (v/v) formamide plus 0.1% bovine serum albumin/0.1% Ficoll/0.1% polydiene pyrrolidone/50 mM sodium phosphate buffer pH 6.5 at 42℃and 750mM sodium chloride, 75mM sodium citrate; or (3) hybridization overnight at 42℃with 50% formamide, 5 XSSC (0.75M sodium chloride, 0.075M sodium citrate 1), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50.mg/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 further high stringency washing in 0.1 XSSC with EDTA at 55 ℃. Moderately stringent conditions may be as described in Sambrook et al, molecular Cloning: a Laboratory Manual, new York: cold Spring Harbor Press, 1989. Medium stringency conditions include the use of wash solutions and hybridization conditions (e.g., temperature, ionic strength, and SDS percentage) that are less stringent than those described above. For example, moderately stringent conditions include hybridization with at least about 16% v/v to at least about 30% v/v formamide and at least about 0.5M to at least about 0.9M salt at 42℃and washing with at least about 0.1M to at least about 0.2M salt at 55 ℃. Moderately stringent conditions can also include the use of 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5M NaHPO 4 (pH 7.2), 7% SDS at 65℃and (i) 2 XSSC, 0.1% SDS; or (ii) 0.5% BSA, 1mM EDTA, 40mM NaHPO 4 (pH 47.2)、5% SDS was washed at 60-65 ℃. The practitioner will adjust the temperature, ionic strength, etc. based on factors such as probe length. The stringency at which nucleic acids hybridize 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 hybrids containing these sequences. The relative stability of nucleic acid hybridization (corresponding to higher Tm) decreases in the following order: RNA, DNA, RNA and 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 herein have codon optimized the sequence encoding the mature protein based on the mdlA gene derived from Penicilliurn camembertii U-150, based on the codon preference of pichia pastoris, and the synthesized gene expressed using pichia pastoris GS115 or mc1-1H as the expression host.
In a specific embodiment, the present application provides a nucleotide sequence (SEQ ID NO: 7) encoding a mature peptide of a monoglyceride-diglyceride lipase (SEQ ID NO: 8) obtained by codon optimization.
The nucleotide sequence of SEQ ID NO. 7 is shown below:
GATGTCTCCACTTCCGAACTGGACCAGTTCGAGTTCTGGGTTCAATACGCAGCCGCCTCTTACTACGAGGCTGATTACACCGCACAGGTTGGTGATAAGCTGTCCTGCTCTAAGGGTAACTGCCCAGAAGTTGAAGCAACCGGTGCAACTGTGTCTTACGACTTCTCCGATTCCACGATCACTGACACCGCAGGTTACATCGCAGTTGATCACACCAACTCCGCAGTGGTACTGGCATTCCGTGGTTCTTACTCCGTACGTAACTGGGTTGCTGATGCTACTTTCGTCCATACCAACCCAGGTCTGTGTGATGGTTGTCTGGCTGAGCTGGGTTTCTGGTCTTCCTGGAAGCTGGTTCGTGATGATATTATCAAAGAACTGAAAGAAGTGGTGGCACAGAACCCAAACTATGAACTGGTGGTCGTGGGCCACTCCCTGGGTGCTGCTGTGGCTACTCTGGCTGCTACCGACCTGCGTGGTAAAGGTTATCCATCTGCTAAACTGTACGCTTACGCTTCCCCTCGTGTTGGCAACGCAGCCCTGGCCAAATATATCACCGCCCAGGGCAACAACTTCCGTTTCACCCACACCAATGACCCAGTACCTAAACTGCCACTGCTGTCTATGGGCTATGTACATGTTTCTCCTGAATATTGGATCACCTCTCCTAACAACGCCACTGTTTCTACCTCTGACATCAAAGTCATTGACGGCGACGTATCTTTTGACGGCAATACCGGCACGGGCCTGCCTCTGCTGACGGACTTTGAAGCCCACATTTGGTACTTTGTACAGGTTGACGCCGGCAAAGGTCCTGGCCTGCCATTCAAACGTGTTTAA
the amino acid sequence of SEQ ID NO. 8 is shown below:
DVSTSELDQFEFWVQYAAASYYEADYTAQVGDKLSCSKGNCPEVEATGATVSYDFSDSTITDTAGYIAVDHTNSAVVLAFRGSYSVRNWVADATFVHTNPGLCDGCLAELGFWSSWKLVRDDIIKELKEVVAQNPNYELVVVGHSLGAAVATLAATDLRGKGYPSAKLYAYASPRVGNAALAKYITAQGNNFRFTHTNDPVPKLPLLSMGYVHVSPEYWITSPNNATVSTSDIKVIDGDVSFDGNTGTGLPLLTDFEAHIWYFVQVDAGKGPGLPFKRV
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 total lengths may vary significantly. It is therefore contemplated that polynucleotide fragments of nearly 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 their fusions may be prepared, manipulated and/or expressed using any of a variety of maturation techniques known and available in the art. For example, polynucleotide sequences encoding the polypeptides of the present application, or variants thereof, may be used in recombinant DNA molecules to direct expression of the polypeptides in an appropriate host cell. Because of the degeneracy inherent in 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 cloning, processing, expression, and/or alteration of activity of gene products.
In certain embodiments, polynucleotides of the present application are produced by artificial synthesis, e.g., 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 in the present application is derived from Penicillium carpentry (Penicilliurn camembertii) U-150 (GeneBank: BAA 14345.1), the codon is optimized according to the codon preference of Pichia pastoris, the optimized gene is synthesized by the company Shanghai, inc., and the synthesized gene is ligated into pUC 57.
In certain embodiments, the sequence of the obtained polynucleotide may be determined by conventional methods, preferably by 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.
Expression vector
The present application provides expression vectors comprising the polynucleotides of the present application.
An "expression vector" as described herein is a recombinantly or synthetically produced nucleic acid construct having a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector used in the present application may be a plasmid vector such as pPIC9K, pUC, pET-24a (+), pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315, or a viral vector such as 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 polypeptides set forth in any one of SEQ ID NOs 1-3 and 15 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 pPIC9K or pUC57.
In particular embodiments, the above-described expression vectors further comprise a regulatory sequence that regulates the expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence.
The term "regulatory sequence" as used herein refers to a polynucleotide sequence required to effect expression of a coding sequence to which it is linked. The nature of such regulatory sequences varies with the host organism. In prokaryotes, such regulatory sequences typically include promoters, ribosome binding sites and terminators; in eukaryotes, such regulatory sequences typically include promoters, terminators and, in some cases, enhancers. Thus, the term "regulatory sequence" includes all sequences whose presence is minimal necessary for expression of the gene of interest, but may also include other sequences whose presence is advantageous for expression of the gene of interest, such as a leader sequence.
The term "operably linked" as used herein refers to the following circumstance: 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 allows expression of the coding sequence under conditions compatible with the regulatory sequence.
In certain embodiments, expression vectors comprising nucleotide sequences encoding polypeptides represented by any one of SEQ ID NOs 1-3 and 15, and variants thereof, and suitable transcriptional/translational regulatory elements are constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like (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 the expression vector to direct mRNA synthesis. Representative examples of such promoters include: the lac or trp promoter of E.coli; PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, LTRs from retroviruses, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. Expression vectors also include ribosome binding sites for translation initiation, transcription terminators, and the like. Insertion of the enhancer sequence into the vector will result in enhanced transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA expression, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer 100 to 270 base pairs late in the replication origin, the polyoma enhancer late in the replication origin, and adenovirus enhancers, among others.
In addition, the expression vector preferably comprises 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 cells
The present application provides host cells comprising the polynucleotides or expression vectors disclosed herein.
In certain embodiments, polynucleotides encoding the polypeptides and variants thereof set forth in any one of SEQ ID NOs 1-3 and 15 or expression vectors containing the polynucleotides are transformed or transduced into host cells to obtain genetically engineered host cells containing the polynucleotides or expression vectors.
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, bacillus subtilis, bacillus cereus, salmonella typhimurium, pseudomonas fluorescens. Exemplary fungal cells include any of the species Aspergillus. Exemplary yeast cells include any of the genera pichia, lager, schizosaccharomyces, or saccharomyces, including pichia, lager, or schizosaccharomyces. Exemplary insect cells include any of the species 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. The selection of an appropriate host is within the ability of those skilled in the art.
In a specific embodiment, the host cell used herein is E.coli. In a preferred embodiment, an expression vector carrying a polynucleotide sequence of the present application is transformed into E.coli DH 5. Alpha. Strain for inducible expression. In another specific embodiment, an expression vector carrying a polynucleotide sequence of the present application is transformed into pichia cells for expression. Pichia pastoris useful in the present application includes GS115 (purchased from American Type Culture Collection (ATCC)), mc1-1H (which was deposited by the applicant of the present application at China general microbiological culture Collection center (CGMCC, address: institute of microorganisms, national academy of sciences of China, no. 3, north Chen West Lu 1, the area of Korea, beijing, and post code: 100101), and has classification designations Pichia pastoris, accession number CGMCC 16668 and KM71 (purchased from American Type Culture Collection (ATCC)), all of which have HIS4 auxotroph markers. GS115 strain has AOX1 gene, is mut+, i.e. normal type of methanol utilization; the mc1-1H strain has an AOX1 gene and is mut+, namely normal methanol utilization; the AOX1 locus of KM71 strain is inserted by ARG4 gene, the phenotype is Muts, namely slow methanol utilization, and all three strains are suitable for a general yeast transformation method.
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. Specific methods include calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection or electroporation, and the like (Davis, L., dibner, M., battey, I., basic Methods in Molecular Biology, (1986)). As an example, when the host is a prokaryote such as E.coli, competent cells can be harvested after the exponential growth phase using CaCl as is well known in the art 2 The method is used for transformation.
In a specific embodiment, recombinant expression vectors containing polynucleotides encoding MDGL mutants are digested and linearized with SalI restriction enzymes, and then transformed into Pichia pastoris GS115 or mc1-1H competent cells by electric shock, and plated onto selection media MGYS screening plates for culture. And (3) picking the transformant to culture on a glycerol dioleate flat plate, and finally picking the transformant with the largest hydrolysis ring for shake flask fermentation test.
Screening and preparation method of polypeptide
The polypeptides of the present application may be screened and prepared by any suitable method known to those of skill in the art.
In some embodiments, the polypeptides disclosed herein are obtained by site-directed mutagenesis of MDGL, and the results can be validated and also used to direct directed engineering of MDGL.
In some embodiments, the polypeptides of the present application may also be produced by recombinant techniques, or chemically synthesized. Methods for producing recombinant peptides are known in the art. Methods of chemical synthesis of peptides are also well known to those skilled in the art, and for example, 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 may be performed manually or by automation. For example, automated synthesis can be accomplished using a 431A peptide synthesizer (Perkin Elmer) of Applied Biosystems. Alternatively, the different fractions may be synthesized chemically and combined chemically, respectively, to produce the desired molecule.
In a specific embodiment, the mdlA gene is subjected to site-directed mutagenesis using a rapid recombination cloning method, and PCR and recombination are performed using TOYOBO KOD enzyme and primers to obtain a plasmid containing the mutated sequence. The resulting fragment is cloned into a plasmid vector, e.g., pPIC9K, via avrili and EcoRI cleavage sites, and the resulting vector is transformed into a host cell, e.g., e. After the mutant is cultured in a medium such as ampicillin, the plasmid is extracted, linearized with SalI, and the amplified recombinant vector (e.g., a fragment of about 10 kb) is recovered and then introduced into yeast cells. The yeast cells into which the recombinant vector is introduced are cultured on a screening medium plate, and mutants with high enzyme activity and/or high heat stability are screened by measuring the enzyme activity of the supernatant medium.
In some embodiments, the selected mutants may also be validated, e.g., fermentation validated. In some embodiments, the validated mutants are sequenced to determine the sequence after mutation.
In some embodiments, the mutated sequences obtained by screening are cloned into vectors, e.g., plasmid vectors, and then introduced into yeast cells, e.g., transformed into competent cells of pichia pastoris GS115 or mc1-1H strain by electrotransformation, and fermentation to verify the enzymatic activity and thermostability of the polypeptide.
In some embodiments, a method of screening for a polypeptide disclosed herein comprises:
1) Mutating the nucleotide sequence shown in SEQ ID NO. 7 to obtain a mutant sequence;
2) Expressing the mutated sequence obtained in step 1) to obtain a polypeptide mutated with respect to the polypeptide shown in SEQ ID NO. 8; and
3) Mutant polypeptides with enzyme activity and/or thermal stability higher than those of the polypeptide shown in SEQ ID NO. 8 are screened.
In certain embodiments, the mutation performed in the above method on the nucleotide sequence set forth in SEQ ID NO. 7 is a site-directed mutation.
In certain embodiments, the expression described in the above methods comprises transforming an expression vector comprising the mutant sequence into a yeast strain. In a specific embodiment, the expression comprises cloning the mutated sequence obtained in step 1) onto an expression vector, and then transforming or transducing into a suitable host cell; and culturing the host cell, recovering the recombinant expression vector, and then transforming the product into a yeast strain for culturing.
In certain embodiments, the mutation site of the mutant polypeptide is selected from any one or a combination of the following: 22, 25, 27, 84, 116, 136, 209, 229, 230, 231, 260, and 269.
In certain embodiments, the mutant polypeptide comprises any one or a combination of the following mutation sites: Y22C, D25R, T R, Y H/R, W116A/H/F, N136D, M209A/V, S229R, T230R, S231R, I E/R/H and G269C.
In certain embodiments, the mutation site comprises Y22C and G269C. In certain embodiments, the mutation site comprises S229R, T230R and S231R. In certain embodiments, the mutation site comprises Y22C, S229R, T230R, S R and G269C. In certain embodiments, the mutation site comprises Y22C, T27R, Y H, N35136D, S229R, T230R, S231R and G269C.
In a specific embodiment, the mutation sites of MDGLs (SEQ ID NO: 1) are Y22C and G269C. In a specific embodiment, the mutation sites of MDGLc (SEQ ID NO: 2) are S229R, T230R and S231R. In a specific embodiment, the mutation sites of MDGLsc (SEQ ID NO: 3) are Y22C, S229R, T230R, S231R and G269C. In a specific embodiment, the mutation sites of MDGL-3G3 (SEQ ID NO: 15) are Y22C, T27R, Y84H, N136D, S229R, T230R, S R and G269C.
In exemplary embodiments, the method of screening for a polypeptide of the present application comprises one or more of the following steps:
1) Mutating the nucleotide sequence shown in SEQ ID NO. 7 to obtain a mutant sequence;
2) Cloning the mutated sequence obtained in step 1) onto an expression vector and then transforming or transducing into a suitable host cell;
3) Culturing the host cell in step 2), recovering the recombinant expression vector, and then converting the product into a yeast strain for culturing; and
4) Mutant polypeptides with enzyme activity and/or thermal stability higher than those of the polypeptide shown in SEQ ID NO. 8 are screened.
Suitable host cells are those suitable for expression of the expression vector or polynucleotide of interest. Suitable media refer to media suitable for growth or inducible expression of the host cell.
In certain embodiments, various conventional media may be selected depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. Preferably, the engineered host cells may be cultured in conventional nutrient media modified as appropriate for activating promoters to screen transformants or amplify the polynucleotides of the present application. After transformation of the appropriate host cell and growth of the host cell to the appropriate cell density, the selected promoter is induced by a suitable method (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 fragment thereof.
In certain embodiments, the polypeptide produced by the host cell may be coated in the cell, or expressed on a cell membrane, or secreted out of the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. For example, the expressed polypeptide or fragment thereof may 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, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
In specific embodiments, the polypeptides disclosed herein are secreted into the medium outside the host cell, e.g., outside the yeast cell. In some embodiments, the cultured bacterial fluid is centrifuged and the supernatant is taken for use in determining the activity of the polypeptide, e.g., the ability to hydrolyze pNPP.
Use of polypeptides having monoglyceride-diglyceride lipase activity
The polypeptide of the application is a polypeptide with lipase activity, in particular a polypeptide with glycerol mono-diacyl ester lipase (MDGL) activity, and can utilize esterification or transesterification reaction to produce glycerol monoacyl ester (MAG) with higher industrial value.
The polypeptides disclosed herein have higher enzyme activity and/or thermostability. In some embodiments, the polypeptide disclosed herein (e.g., a polypeptide set forth in any one of SEQ ID NOS: 1-3 and 15) has a higher pNPP hydrolysis activity than wild-type MDGL (e.g., a control polypeptide set forth in SEQ ID NO: 8). In some embodiments, the polypeptides disclosed herein (e.g., the polypeptides set forth in SEQ ID NO:1, 3, or 15) have a thermostability that is superior to wild-type MDGL. In some embodiments, the polypeptides disclosed herein (e.g., the polypeptides shown in SEQ ID NO:2, 3, or 15) have a higher specific enzymatic activity than wild-type MDGL (e.g., the control polypeptide shown in SEQ ID NO: 8). In some embodiments, the MDGL mutants disclosed herein (e.g., the polypeptides shown in SEQ ID NO:3 or 15) have significantly higher enzyme activity and thermostability than wild-type MDGL (e.g., the control polypeptides shown in SEQ ID NO: 8).
The polypeptides disclosed herein can be used for MAG synthesis, MAG/DAG hydrolysis, esterification of fatty acids, and MAG/DAG transesterification, among others. The present application provides for the use of the polypeptides disclosed herein to catalyze synthesis, hydrolysis or transesterification reactions of esters, preferably, the esters are monoglycerides and/or diglycerides. 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 "include" 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 particular aspects, embodiments or examples of the present application may be applied to any other aspects, embodiments or examples described herein unless contradicted by context.
The foregoing disclosure generally describes the present application, which is further illustrated by the following examples. These examples are described for illustration only and are not intended to limit the scope of the present application. Although specific terms and values are used herein, these terms and values are likewise to be understood as exemplary and do not limit the scope of the present application.
Examples
Example 1: synthesis and cloning of MDGL sequences
SEQ ID NO:7 (the coding sequence of which is shown as polypeptide 28-3 or MDGL of SEQ ID NO: 8) was synthesized by the division of biological engineering (Shanghai) Co.
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 NEB, and the plasmid was digested with AvrII and EcoRI together with the inducible promoter P AOX1 And the yeast alpha mating factor plasmid pPIC9K, then using Axygen company gel recovery kit purification. The MDGL sequence enzyme fragment and the plasmid enzyme fragment are connected by using T4 DNA ligase of Fermentas company according to the product specification, and the connection heat shock method is transferred into a large scaleIn enterobacteria DH 5. Alpha. Were cultured overnight on LB plates containing ampicillin. The following day, the monoclonals were picked up and cultured in LB liquid medium, plasmids were extracted using the Axygen company plasmid extraction kit and submitted to Shanghai Biotechnology Co.Ltd for sequencing.
The strain with correct sequencing result is used for extracting plasmid, after the plasmid is linearized by SalI, the Pichia pastoris GS115 is transformed by adopting an electric shock method, the transformant is screened on a screening medium without histidine, and only the recombinant Pichia pastoris transformed with exogenous gene fragments can grow on the screening medium. Transferring the colony on the screening culture medium to a BMMY-olive oil screening culture medium plate, selecting the clone according to the size of a hydrolysis circle, and performing shake flask fermentation and activity detection to obtain recombinant Pichia pastoris GS115-MDGL.
Example 2: MDGL site-directed mutagenesis
The plasmid pPIC9K-MDGL containing the polynucleotide encoding MDGL was amplified using the primer pair MDGLm-F1 (CTCTTACTGTGAGGCTGATTACACC, SEQ ID NO: 9) and MDGLm-R1 (CCTTTGCAGGCGTCAACCTGTACAAAGTACC, SEQ ID NO: 10), and the primer pair MDGLm-F2 (TTGACGCCTGCAAAGGTCCTGGCCT, SEQ ID NO: 11) and MDGLm-R2 (CAGCCTCACAGTAAGAGGCGGCTGC, SEQ ID NO: 12), and PCR was performed using the KOD enzyme of TOYOBO Co., as follows: mu.L of a 25. Mu. L, dNTP mixture of 2 Xbuffer (2.5 mM each), 1.5. Mu.L of each primer, 1. Mu.L of plasmid template 1. Mu. L, KOD enzyme, and double distilled water was added to 50. Mu.L. The PCR reaction procedure was as follows: 98℃for 10s, 56℃for 20s, 68℃for 1kb/min, 68℃for 5min,30 cycles. The PCR product was purified using the PCR purification kit from Axygen. The purified PCR products were ligated by using a rapid recombination kit (Tiangen Biochemical technology (Beijing) Co., ltd.), the resulting vector was transformed into E.coli DH 5. Alpha. Strain, the positive clones were sequenced, the strain containing the correct mutated sequence was inoculated into LB medium containing 100mg/mL ampicillin, cultured at 37℃for 16 hours, and plasmids were extracted using an Axygen Minipre plasmid extraction kit to obtain plasmid pPIC9K-MDGLs containing the mutated sequence. The nucleotide sequence of the mutant sequence is shown as SEQ ID NO:4, the coded MDGL mutant MDGLs has an amino acid sequence shown in SEQ ID NO: 1.
The plasmid pPIC9K-MDGL containing the polynucleotide encoding MDGL was amplified using primer pairs MDGL-ctlm-F (CGTAGACGTGACATCAAAGTCATTGACGGCG, SEQ ID NO: 13) and MDGL-ctlm-R (GATGTCACGTCTACGAACAGTGGCGTTGTTAGGAGAGG, SEQ ID NO: 14), and PCR was performed using KOD enzyme from TOYOBO Co., ltd. In the following reaction system: mu.L of a 25. Mu. L, dNTP mixture of 2 Xbuffer (2.5 mM each), 1.5. Mu.L of each primer, 1. Mu.L of plasmid template 1. Mu. L, KOD enzyme, and double distilled water was added to 50. Mu.L. The PCR reaction procedure was as follows: 98℃for 10s, 56℃for 20s, 68℃for 10min, 68℃for 5min,30 cycles. The PCR product was purified using the PCR purification kit from Axygen. The purified PCR products were ligated by using a rapid recombination kit (Tiangen Biochemical technology (Beijing) Co., ltd.), the resulting vector was transformed into E.coli DH 5. Alpha. Strain, the positive clones were sequenced, the strain containing the correct mutated sequence was inoculated into LB medium containing 100mg/mL ampicillin, cultured at 37℃for 16 hours, and plasmids were extracted using an Axygen Minipre plasmid extraction kit to obtain plasmid pPIC9K-MDGLc containing the mutated sequence. The nucleotide sequence of the mutant sequence is shown as SEQ ID NO:5, the coded MDGL mutant MDGLc has an amino acid sequence shown in SEQ ID NO: 2.
The plasmid pPIC9K-MDGLs containing the polynucleotide encoding MDGLs was amplified using primer pairs MDGL-ctlm-F and MDGL-ctlm-R, and PCR was performed using KOD enzyme from TOYOBO Co., ltd. The reaction system is as follows: mu.L of a 25. Mu. L, dNTP mixture of 2 Xbuffer (2.5 mM each), 1.5. Mu.L of each primer, 1. Mu.L of plasmid template 1. Mu. L, kKOD enzyme, and double distilled water was added to 50ul. The PCR reaction procedure was as follows: 98℃for 10s, 56℃for 20s, 68℃for 10min, 68℃for 5min,30 cycles. The PCR product was purified using the PCR purification kit from Axygen. The purified PCR products were ligated by using a rapid recombination kit (Tiangen Biochemical technology (Beijing) Co., ltd.), the resulting vector was transformed into E.coli DH 5. Alpha. Strain, the positive clones were sequenced, the strain containing the correct mutated sequence was inoculated into LB medium containing 100mg/mL ampicillin, cultured at 37℃for 16 hours, and plasmids were extracted using an Axygen Minipre plasmid extraction kit to obtain plasmid pPIC9K-MDGLsc containing the mutated sequence. The nucleotide sequence of the mutant sequence is shown as SEQ ID NO:6, the coded MDGL mutant MDGLsc has an amino acid sequence shown in SEQ ID NO: 3.
Example 3: mutant transformed pichia pastoris and panel screening
Recombinant expression vectors containing polynucleotides encoding MDGL mutants (MDGLs, MDGLc or MDGLsc) and wild type MDGL (polypeptide 28-3) which were sequenced correctly were digested and linearized with SalI restriction enzyme, and then transformed into Pichia pastoris GS115 competent cells by electric shock, plated onto selection medium MGYS screening plates, and cultured at 28℃for three days. Transformants were picked up on glycerol dioleate plates (1% YNB, 2% phospholipid, 2% agarose, 2% methanol, 0.5% glycerol dioleate) and incubated at 28℃for 1 day, and transformants with the largest hydrolytic circles were picked up for shake flask fermentation testing.
Example 4: fermentation verification of strain producing monoglyceride-diacyl ester lipase
The larger transparent circle of the glycerol dioleate plate of example 3 was picked up and cultured in 50mL BMGY medium (1% yeast extract, 2% peptone, 1.34% ammonium sulfate-containing yeast nitrogen source base (YNB) without amino acid, 2% glycerol, 4X 10-5% D-biotin, 100mM citric acid-sodium citrate buffer, pH 6.6), 28℃and 200rpm for 1 day, and OD was measured 600 Taking a certain volume of culture solution, centrifuging at 8000rpm for 2 min, and re-suspending into 250mL triangular flask containing 50mL BMMY (1% yeast extract, 2% peptone, 1.34% yeast nitrogen source alkali (YNB) containing ammonium sulfate and no amino acid, 2% methanol, 4×10-5% D-biotin, 100mM citric acid-sodium citrate buffer solution, pH 6.6), and the initial bacterial liquid OD 600 The culture was carried out at 28℃and 200rpm, 500. Mu.L of methanol was added every 12 hours, and the culture was carried out for 3 days. After completion of the culture, all the bacterial solutions were collected, centrifuged at 8000rpm for 5 minutes, and the total supernatant was collected and ultrafiltered with a 10K filter membrane (Millipore) and the protein concentration was measured using a Bradford reagent (manufactured and bioengineered (Shanghai) Co., ltd.). The protein concentrations of MDGLs, MDGLc, MDGLsc and 28-3 were 0.16mg/mL, 0.13mg/mL and 0.45mg/mL, respectively, as shown in FIG. 1.
Example 5: enzyme activity yield of MDGL detected by pNPP method
To 400. Mu.L of the pNPP reaction solution (3 mg/mL, dissolved in isopropyl alcohol, 1mL was mixed with 9mL of a 0.2M sodium acetate-acetic acid solution before use), 5. Mu.L of the enzyme solution was added, the reaction was continued at 37℃for 20 minutes at 1000rpm, 400. Mu.L of a stop solution (200 mM Tris-HCl, 5% triton-100, pH 8.5) was added, the reaction was stopped, centrifugation was performed at 12000rpm for 5 minutes, and the supernatant was collected and the absorbance at 405nm was measured.
Enzyme activity unit=0.1935 od 405 *V 1 * Dilution factor/T/V 2
V 1 : volume of reaction solution (mL), V 2 : enzyme volume (mL), T: time (min).
The measurement results were as follows: 28-3 was 7.3U/mL, MDGLs was 2.5U/mL, MDGLc was 0.9U/mL, and MDGLsc was 1U/mL, see FIG. 2.
Example 6: MDGL mutant thermal stability test
Taking 50 mu L of fermentation supernatant, preserving heat at 40-50 ℃ for 30 minutes, immediately measuring pNPP hydrolysis capacity after the heat preservation is finished, and calculating enzyme activity residual rate and enzyme activity residual rate by taking the supernatant which is not subjected to heat treatment as a referenceThe results show that, compared to polypeptide 28-3, MDGLs is +.>About 1℃increase in MDGLsc +.>An increase of about 2 ℃ (fig. 3), which indicates an increase in the thermal stability of MDGLs and MDGLsc, especially an increase in the thermal stability of MDGLsc is more pronounced.
Example 7: SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of MDGL mutant
In order to exclude the effect of the impurity proteins, 3 mutants (MDGLs, MDGLc and MDGLsc) and wild type MDGL (28-3) were subjected to SDS-PAGE analysis (FIG. 4), all samples were protein with pNPP hydrolysis activity of 16U, the band of MDGLs was thicker than that of wild type MDGL, which indicated that the specific enzyme activity of MDGLs was reduced, the band of MDGLc was significantly thinner than that of wild type MDGL, which indicated that the specific enzyme activity of MDGLc was significantly improved, and the band of MDGLsc was thinner than that of wild type MDGL, which indicated that the specific enzyme activity of MDGLsc was improved.
Example 8: synthesis and cloning of MDGL-3G3 sequence
SEQ ID NO:16 (the coding sequence of which is shown as the polypeptide MDGL-3G3 shown in SEQ ID NO: 15) is synthesized by the division of biological engineering (Shanghai) Co.
The synthesized MDGL-3G3 sequence was cloned in plasmid pUC57, stored in E.coli DH 5. Alpha. And the plasmid was digested with AvrII and EcoRI from NEB, and the plasmid was digested with AvrII and EcoRI together with the inducible promoter P AOX1 And the yeast alpha mating factor plasmid pPIC9K, then using Axygen company gel recovery kit purification. The MDGL-3G3 sequence fragment and the plasmid enzyme fragment were ligated using Fermentas T4 DNA ligase according to the product instructions, and the ligation was transferred into E.coli DH 5. Alpha. By heat shock and cultured overnight on LB plates containing ampicillin. The following day, the monoclonals were picked up and cultured in LB liquid medium, plasmids were extracted using the Axygen company plasmid extraction kit and submitted to Shanghai Biotechnology Co.Ltd for sequencing.
Example 9: transformation of Pichia pastoris and Panel screening with the mutant MDGL-3G3
The recombinant expression vector containing the polynucleotide for encoding MDGL mutant (MDGL-3G 3) with correct sequencing result is digested and linearized by SalI restriction enzyme, and then pichia pastoris mc1-1H competent cells are transformed by adopting an electric shock method, spread on a selection medium MGYS screening plate and cultured for three days at 28 ℃. Transformants were picked up on glycerol dioleate plates (1% YNB, 2% phospholipid, 2% agarose, 2% methanol, 0.5% glycerol dioleate) and incubated at 28℃for 1 day, and transformants with the largest hydrolytic circles were picked up for shake flask fermentation testing. The control strain was sequence-optimized MDGL with no mutation in amino acid sequence.
Example 10: fermentation verification of strain producing monoglyceride-diacyl ester lipase
5 large transparent circles of the glycerol dioleate plates of example 9 were picked up and placed in 50mL BMGY medium (1% yeast extract, 2% peptone, 1.34% ammonium sulfate-containing and amino acid-free yeast nitrogen source base (YNB), 2% glycerol, 4X 1)0-5% D-biotin, 100mM citric acid-sodium citrate buffer, pH 6.6), 28℃and 200rpm for 1 day, and OD was measured 600 . The culture broth was centrifuged at 8000rpm for 2 minutes and resuspended in a 250mL Erlenmeyer flask containing 50mL BMMY (1% yeast extract, 2% peptone, 1.34% Yeast Nitrogen Base (YNB) with ammonium sulfate without amino acids, 2% methanol, 4X 10-5% D-biotin, 100mM citric acid-sodium citrate buffer, pH 6.6), incubated at 28℃at 200rpm with 500. Mu.L of methanol added every 12h for 3 days. After completion of the culture, all the bacterial solutions were collected, centrifuged at 8000rpm for 5 minutes, and the total supernatant was collected and ultrafiltered with a 10K filter membrane (Millipore) and the protein concentration was measured using a Bradford reagent (manufactured and bioengineered (Shanghai) Co., ltd.). The protein concentration of MDGL-3G3-1 to MDGL-3G3-5 was 0.22-0.64mg/mL, and the protein yield of the control strain was 0.69mg/mL, see FIG. 5.
Example 11: detection of enzyme activity yield of MDGL-3G3 mutant by pNPP method
To 400. Mu.L of the pNPP reaction solution (3 mg/mL, dissolved in isopropyl alcohol, 1mL was mixed with 9mL of a 0.2M sodium acetate-acetic acid solution before use), 5. Mu.L of the enzyme solution was added, the reaction was carried out at 37℃for 10 minutes at 1000rpm, 400. Mu.L of a stop solution (200 mM Tris-HCl, 5% triton-100, pH 8.5) was added to terminate the reaction, centrifugation was carried out at 12000rpm for 5 minutes, and the supernatant was collected and the absorbance at 405nm was measured.
Enzyme activity unit=0.1935 od 405 *V 1 * Dilution factor/T/V 2
V 1 : volume of reaction solution (mL), V 2 : enzyme volume (mL), T: time (min).
Specific enzyme activity = enzyme activity yield/protein concentration
The measurement results were as follows: the enzyme activity yield of the wild MDGL is 14.1U/mL, the specific enzyme activity is 20.51U/mg, the enzyme activity yield of the MDGL-3G3-2 is 25.1U/mL, the specific enzyme activity is 43.1U/mg, and the specific enzyme activity is improved by 110.1% compared with the wild MDGL; the enzyme activity yield of MDGL-3G3-3 is 19.0U/mL, the specific enzyme activity is 29.9U/mg, and the specific enzyme activity is improved by 45.9% compared with that of the wild MDGL, see FIG. 6.
Example 12: MDGL-3G3 mutant thermal stability test
Taking 50 mu L of each of the MDGL-3G3-3 and MDGL fermentation liquid, preserving the temperature at 37-62 ℃ for 30 minutes, immediately measuring the pNPP hydrolysis capability after the heat preservation, and calculating the enzyme activity residual rate by taking the supernatant which is not subjected to heat treatment as a reference. As a result, the residual activity ratio of MDGL-3G3 after heat preservation treatment at 41.8-52.2 ℃ for 30min is obviously higher than that of MDGL, which shows that the heat stability of MDGL-3G3 is obviously improved, as shown in FIG. 7.
Example 13: SDS-PAGE analysis of MDGL-3G3 mutant and MDGL (sodium dodecyl sulfate polyacrylamide gel electrophoresis)
A large amount of non-target proteins are present in the fermentation broth, and SDS-PAGE analysis is performed on two mutants of MDGL-3G3-2 and MDGL-3G3-3 and wild type MDGL (FIG. 8), and all samples are proteins with pNPP hydrolysis activity of 150U, and the bands of MDGL-3G3-2 and MDGL-3G3-3 are finer than that of the wild type MDGL, which indicates that the specific enzyme activities of MDGL-3G3-2 and MDGL-3G3 are improved.
It should be understood that while the application is illustrated in some form, the application is not limited to what has been shown and described in this specification. It will be apparent to those skilled in the art that various changes can be made without departing from the scope of the application. Such variations are within the scope of the claims of the present application.
Sequence listing
<110> Feng Yi (Shanghai) Biotechnology research and development center Co., ltd
<120> novel monoglyceride-diacyl ester lipase
<150> CN 201911329804.5
<151> 2019-12-20
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Ala Ala Ala Ser Tyr Cys Glu Ala Asp Tyr Thr Ala Gln Val Gly Asp
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Ala Thr Val Ser Tyr Asp Phe Ser Asp Ser Thr Ile Thr Asp Thr Ala
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gatgtctcca cttccgaact ggaccagttc gagttctggg ttcaatacgc agccgcctct 60
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gtcgtgggcc actccctggg tgctgctgtg gctactctgg ctgctaccga cctgcgtggt 480
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gtacctaaac tgccactgct gtctatgggc tatgtacatg tttctcctga atattggatc 660
acctctccta acaacgccac tgtttctacc tctgacatca aagtcattga cggcgacgta 720
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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
<210> 9
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ctcttactgt gaggctgatt acacc 25
<210> 10
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
cctttgcagg cgtcaacctg tacaaagtac c 31
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ttgacgcctg caaaggtcct ggcct 25
<210> 12
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
cagcctcaca gtaagaggcg gctgc 25
<210> 13
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
cgtagacgtg acatcaaagt cattgacggc g 31
<210> 14
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gatgtcacgt ctacgaacag tggcgttgtt aggagagg 38
<210> 15
<211> 279
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
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 Cys Glu Ala Asp Tyr Arg 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 Arg Arg Arg 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 Cys Lys Gly Pro
260 265 270
Gly Leu Pro Phe Lys Arg Val
275
<210> 16
<211> 840
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gacgtttcta cttccgagtt ggaccaattc gagttctggg ttcaatacgc tgctgcttct 60
tactgtgagg ctgattacag agctcaagtt ggagataagt tgtcttgttc taagggtaac 120
tgtcctgaag ttgaggctac tggtgctact gtttcttacg atttctccga ttctactatc 180
actgatactg ccggttacat tgctgttgat cacactaact ctgctgttgt tttggctttc 240
agaggttctc actctgttcg taactgggtt gctgacgcta cttttgttca cactaaccca 300
ggtttgtgtg atggttgttt ggctgagttg ggtttctggt cctcctggaa gttggttaga 360
gatgacatta ttaaggagtt gaaggaggtt gtcgctcaaa accctgacta cgagttggtt 420
gttgttggtc actctttggg tgctgccgtt gctaccttgg ctgccactga cttgagaggt 480
aaaggttacc catctgctaa gttgtacgct tacgcttctc caagagttgg taacgctgct 540
ttggctaagt acattaccgc tcaaggtaac aacttcagat tcactcacac taacgatcca 600
gttccaaagt tgccattgtt gtccatgggt tacgttcacg tttctccaga gtactggatt 660
acttccccaa acaacgctac tgttcgtaga cgtgacatta aggttatcga tggagatgtt 720
tctttcgacg gtaacactgg taccggtttg ccattgttga ctgacttcga ggctcacatc 780
tggtacttcg ttcaagttga tgcttgtaag ggtccaggtt tgccattcaa gagagtttaa 840

Claims (10)

1. A polypeptide having glycerol mono-diacyl ester lipase activity, which consists of an amino acid sequence shown in any one of SEQ ID NOs 1 to 3 and 15.
2. A polynucleotide encoding the polypeptide of claim 1.
3. The polynucleotide according to claim 2, which consists of the nucleotide sequence shown in any one of SEQ ID NOs 4 to 6 and 16.
4. An expression vector comprising at least one polynucleotide of claim 2 or 3.
5. The expression vector of claim 4, further comprising a regulatory sequence that regulates expression of the polynucleotide, wherein the polynucleotide is operably linked to the regulatory sequence.
6. The expression vector of claim 5, wherein the expression vector is a plasmid.
7. A host cell comprising the polynucleotide of claim 2 or 3, or the expression vector of any one of claims 4-6.
8. The host cell of claim 7, wherein the host cell is yeast or e.
9. Use of the polypeptide of claim 1 for catalyzing synthesis, hydrolysis or transesterification of monoglycerides and/or diglycerides.
10. A method of synthesizing a monoglyceride and/or diglyceride comprising contacting the polypeptide of claim 1 with a fatty acid and glycerol.
CN202011499617.4A 2019-12-20 2020-12-17 Novel glycerol mono-diacyl ester lipase Active CN113005111B (en)

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CN112574975B (en) * 2020-09-30 2022-04-01 华南理工大学 Glyceride lipase mutant G28C-P206C, and coding gene and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811449A (en) * 2015-11-27 2017-06-09 丰益(上海)生物技术研发中心有限公司 A kind of new lipase B M19
CN108251400A (en) * 2016-12-29 2018-07-06 丰益(上海)生物技术研发中心有限公司 Lipase and its application
CN109929820A (en) * 2017-12-19 2019-06-25 丰益(上海)生物技术研发中心有限公司 Novel Mono-and diacylglycerol lipase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811449A (en) * 2015-11-27 2017-06-09 丰益(上海)生物技术研发中心有限公司 A kind of new lipase B M19
CN108251400A (en) * 2016-12-29 2018-07-06 丰益(上海)生物技术研发中心有限公司 Lipase and its application
CN109929820A (en) * 2017-12-19 2019-06-25 丰益(上海)生物技术研发中心有限公司 Novel Mono-and diacylglycerol lipase

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