WO2020135657A1 - 一种高酶活的磷脂酶c突变体 - Google Patents

一种高酶活的磷脂酶c突变体 Download PDF

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WO2020135657A1
WO2020135657A1 PCT/CN2019/128970 CN2019128970W WO2020135657A1 WO 2020135657 A1 WO2020135657 A1 WO 2020135657A1 CN 2019128970 W CN2019128970 W CN 2019128970W WO 2020135657 A1 WO2020135657 A1 WO 2020135657A1
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oil
polypeptide
seq
sequence
amino acid
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PCT/CN2019/128970
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English (en)
French (fr)
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宣姚吉
牛其文
徐正军
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丰益(上海)生物技术研发中心有限公司
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Priority to EP19902338.3A priority Critical patent/EP3904509A4/en
Priority to US17/419,091 priority patent/US20220064611A1/en
Publication of WO2020135657A1 publication Critical patent/WO2020135657A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04003—Phospholipase C (3.1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • the invention relates to a phospholipase C mutant with high enzyme activity.
  • Degumming is an important step in oil refining, and the traditional hydration degumming method has high economic cost, large energy consumption of materials, and heavy environmental pollution. Therefore, in recent years, many efforts have been devoted to the use of enzymatic degumming in the degumming link in oil refining And made great progress. Compared with traditional methods, enzymatic degumming can improve economic efficiency, achieve energy saving and emission reduction, less pollution to the ecological environment, and has greater advantages in environmental protection, economy, and quality.
  • the enzyme used for degumming oils and fats is phospholipase, and compared with other degumming enzymes, phospholipase C (PLC) shows greater advantages, such as increasing the yield of diglyceride (DAG) and reducing the amount of oil obtained loss.
  • BC-PC-PLC Bacillus cereus' PC-PLC
  • BC-PC-PLC Bacillus cereus' PC-PLC
  • the mature peptide is 245 amino acids (Johansen et al. 1988).
  • the crystal structure of BC-PC-PLC has been reported, which is composed of multiple helical domains, the catalytic site is aspartic acid 55, and contains at least three Zn2+ binding sites (Hough et al. 1989).
  • BC-PC-PLC the four amino acids of BC-PC-PLC, namely, asparagine at positions 56, 63, 131, and 134 to histidine, aspartic acid, serine, and aspartic acid, respectively.
  • Aspartic acid the specific enzyme activity is greatly improved compared to the wild type.
  • the inventors selected the 6th, 8th, 10th, 104th, and 205th amino acids in the BC-PC-PLC protein for random saturation mutation , Directed evolution of existing mutants of phospholipase C, hoping to obtain mutants that are further improved than enzyme activity, so as to be able to obtain more efficient phospholipase C, improve degumming efficiency, and increase glycolide (DAG) in the degumming process The yield.
  • DAG glycolide
  • the present invention provides an isolated polypeptide selected from:
  • the 6th amino acid residue is proline or tryptophan; the 8th amino acid residue is alanine, leucine or isoleucine ;
  • the 10th and 104th amino acids are independently serine or threonine.
  • the isolated polypeptide is shown as SEQ ID NO:4.
  • the present invention also provides an isolated polypeptide having an amino acid sequence shown in SEQ ID NO: 4 of at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97 %, more preferably at least 98%, more preferably at least 99% sequence identity, and the amino acid residues at positions 6, 8, 10, and 104 of the isolated polypeptide corresponding to SEQ ID NO: 4 are proline, respectively , Valine, serine and serine; preferably, the polypeptide is obtained from Bacillus subtilis.
  • the invention also provides a polynucleotide sequence selected from:
  • polynucleotide sequence is shown in SEQ ID NO:3.
  • the invention also provides a nucleic acid construct comprising the polynucleotide sequence described in any of the embodiments herein and one or more regulatory sequences operatively linked to the polynucleotide sequence.
  • the nucleic acid construct is a vector.
  • the vector is an expression vector or a cloning vector.
  • the present invention also provides a genetically engineered host cell containing the polynucleotide sequence or nucleic acid construct according to any of the embodiments herein.
  • the present invention also provides a composition containing the polypeptide according to any of the embodiments herein and optional auxiliary materials.
  • the auxiliary materials are selected from activated carbon, alumina, diatomaceous earth, porous ceramics, Absorbing material for porous glass.
  • the present invention also provides the use of the polypeptide, polynucleotide sequence, nucleic acid construct, host cell or composition of any of the embodiments herein in oil refining, phospholipid modification, feed improver, food industry and pharmaceutical industry.
  • the present invention also provides an enzymatic degumming method, the method comprising degumming using the polypeptide according to any of the embodiments herein, preferably, the method comprises the step of contacting the polypeptide with crude oil, preferably, The steps include contacting the polypeptide with 50-70°C crude oil.
  • the method includes one or more of the following features:
  • the added amount of the polypeptide is 10 to 1000 ppm, preferably 50 to 500 ppm, and more preferably 100 to 300 ppm;
  • the degumming includes: stirring at 50 to 60°C for 1 to 3 hours, and then heating to 80 to 90°C for 1 to 10 minutes;
  • the crude oil is selected from: soybean oil, sunflower oil, peanut oil, rapeseed oil, rice bran oil, corn oil, olive oil, palm oil, palm kernel oil, palm soft butter, canola oil, castor oil , coconut oil, coriander oil, cottonseed oil, hazelnut oil, hemp seed oil, linseed oil, mango kernel oil, white mango oil, cattle hoof oil, safflower oil, camellia oil, tall oil, and konjac oil.
  • Figure 1 Specific enzyme activity of the mutant.
  • Figure 3 Standard curve for determination of phospholipase activity.
  • the present invention provides an isolated polypeptide having an amino acid sequence as shown in SEQ ID NO: 7, or at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97 with SEQ ID NO:7 %, more preferably at least 98%, more preferably at least 99% sequence identity, and corresponding to the amino acid residues of at least one of the 6th, 8th, 10th and 104th positions of SEQ ID NO:7
  • the polypeptide is the same as the 6th, 8th, 10th, and/or 104th position of SEQ ID NO: 7, respectively, while retaining the phospholipase C activity of SEQ ID NO: 7.
  • isolated means a form or substance that does not exist in nature.
  • isolated substances include any non-naturally occurring substance and any substance that is at least partially removed from one or more or all naturally occurring components associated with it in nature, including but not limited to any Enzymes, variants, nucleic acids, proteins, peptides or cofactors.
  • the 6th and 8th amino acid residues (Xaa) may be amino acid residues with non-polar side chains, including but not limited to alanine, valine, leucine, iso Leucine, proline, phenylalanine, methionine, tryptophan; amino acid residues (Xaa) at positions 10 and 104 can be amino acids with uncharged polar side chains, including But not limited to glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine.
  • the 6th amino acid residue is proline or tryptophan; the 8th amino acid residue is alanine, leucine or isoleucine; the 10th and The amino acid at position 104 is independently serine or threonine.
  • the amino acid sequence of an exemplary polypeptide is shown in SEQ ID NO:4.
  • the invention also includes one or more (usually 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid mutations based on SEQ ID NO: 7 (Deletion, insertion, and/or substitution), while retaining the phospholipase C activity of the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid mutation is to add one or several (usually within 20, preferably within 10, and more preferably at the C-terminal and/or N-terminal of SEQ ID NO: 7 Within 8) amino acid polypeptide. It should be understood that the one or more amino acid mutations generally do not include the case where all the amino acids (Xaa) at positions 6, 8, 10, and 104 of SEQ ID NO: 7 are mutated.
  • amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains, these families include amino acids with basic side chains (eg, lysine, arginine, histidine), with acidic side Chain amino acids (e.g. aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine Amino acid), amino acids with non-polar side chains (e.g.
  • random saturation mutation refers to designating the site to be mutated in the PCR primer as the NNK merge codon, so as to cover all 20 amino acids to achieve the effect of saturation mutation, and because multiple positions are selected Point mutations are random, and the combination is random, so the author calls this method random mutation mutation.
  • the present invention also includes one or several amino acids in the C-terminus and/or N-terminus of the polypeptide of the present invention or in a suitable region within the protein to facilitate the construction of the polypeptide expression vector, expression of the polypeptide and/or purified amino acids Polypeptides, these polypeptides still have the phospholipase C activity described herein.
  • the present invention includes having at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97 with SEQ ID NO:7 %, more preferably at least 98%, more preferably at least 99% amino acid sequence of sequence identity.
  • the present invention includes having at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 90% with SEQ ID NO:4
  • amino acid residues at the corresponding positions of SEQ ID NO: 4 and 7 remain, for example, at the sixth, 8, 10 and 104 of SEQ ID NO: 4 Proline, valine, serine and serine at the position.
  • amino acid sequence having the sequence identity is obtained from Bacillus subtilis.
  • sequence identity is used to describe the correlation between two amino acid sequences or between two nucleotide sequences. Sequence identity can be calculated using methods well known in the art.
  • the Needleman-Onsch program implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics, 16:276-277) can be used (Needleman-Wunsch) algorithm (Needleman and Wunsch, 1970, Journal of Molecular Biology, 48:443-453) to determine the sequence identity between two amino acid sequences.
  • EMBOSS European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics, 16:276-277
  • Needleman-Wunsch Needleman and Wunsch, 1970, Journal of Molecular Biology, 48:443-453
  • BLASTP on NCBI can be used to calculate sequence identity between two amino acid sequences.
  • polypeptide of the invention may be glycosylated or may be non-glycosylated.
  • the present application includes the nucleotide sequence encoding the polypeptide of the present invention or its complementary sequence.
  • SEQ ID NO: 3 shows an example of the coding sequence of the polypeptide of the present invention.
  • the "coding sequence” includes a nucleic acid sequence encoding the polypeptide of the present invention (especially SEQ ID NO: 7).
  • the sequence encoding the polypeptide of the present invention may be the same as the coding region sequence shown in SEQ ID NO: 3 or a degenerate variant.
  • degenerate variant in the present invention refers to the same amino acid sequence, but the nucleotide sequence has a different nucleotide sequence.
  • the sequence encoding the polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optionally additional coding sequences) and a non-coding sequence.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs, derivatives and variant forms having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is a form of substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the protein it encodes .
  • the invention also includes fragments of the nucleic acid sequence encoding the polypeptide of the invention (eg SEQ ID NO: 3 or its complement).
  • a "nucleic acid fragment” has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to determine and/or isolate polynucleotides encoding the polypeptides of the present invention. Therefore, in some embodiments, the nucleic acid fragments are 15-30 bases in length.
  • the nucleic acid sequence of the present invention can be used to select appropriate nucleic acid fragments for use as primers or probes using existing techniques.
  • the coding sequence of the polypeptide of the present invention or a fragment thereof can generally be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or cDNA prepared according to conventional methods known to those skilled in the art
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the invention also relates to a nucleic acid construct comprising an isolated polynucleotide of the invention operably linked to one or more control sequences expressed in a suitable host cell under conditions suitable for directing the coding sequence in a suitable host cell.
  • operably linked means that the regulatory sequence is located in an appropriate position so as to control and direct the expression of the polynucleotide sequence of interest.
  • the polynucleotide encoding the polypeptide of the present invention can be manipulated in various ways to ensure the expression of the polypeptide.
  • the control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by a host cell for expressing a polynucleotide encoding a polypeptide of the present invention.
  • the promoter sequence contains transcriptional regulatory sequences linked to the expression of the polypeptide.
  • the promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and may be extracellular encoding homologous or heterologous to the host cell Or the gene of the intracellular polypeptide is obtained.
  • suitable promoters for directing the nucleic acid constructs of the present invention, in particular transcription in bacterial host cells are the bacteriophage T7 promoter, E. coli lac operon, Streptomyces coelicolor agarase gene, Promoter sequences obtained from Bacillus subtilis fructan sucrase gene, Bacillus licheniformis alpha-amylase gene, Bacillus amyloliquefaciens alpha-amylase gene, Bacillus licheniformis penicillinase gene, etc.
  • Suitable promoters for directing the forward transcription of the nucleic acid construct of the present invention in filamentous fungal host cells are from Aspergillus oryzae TAKA amylase, Rhizomucor (mihiei) aspartic protease, Aspergillus niger neutral alpha -Amylase, Aspergillus niger-stable alpha-amylase, Aspergillus niger or Aspergillus awamori saccharification enzyme (glaA), Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Aspergillus oryzae alkaline Proteases, Aspergillus oryzae triose phosphate isomerase, Trichoderma reesei endoglucanase and other gene-derived promoters and their mutant, truncated and hybrid promoters.
  • useful promoters can be obtained from Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactose kinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, Saccharomyces cerevisiae Triose phosphate isomerase, Saccharomyces cerevisiae 3-phosphoglycerate kinase gene, Pichia pastoris alcohol oxidase gene.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactose kinase
  • Saccharomyces cerevisiae alcohol dehydrogenase glyceraldehyde 3-phosphate dehydrogenase
  • Saccharomyces cerevisiae Triose phosphate isomerase Saccharomyces cerevis
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3'end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
  • the preferred terminator for the bacterial host may be a terminator from T7 bacteriophage.
  • Preferred terminators for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.
  • Preferred terminators for yeast host cells are obtained from S. cerevisiae enolase, S. cerevisiae cytochrome C, S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase, P. pastoris alcohol oxidase genes, and the like.
  • control sequence may also be a suitable leader sequence, an untranslated region of mRNA important for translation by the host cell.
  • the check-in sequence is operably linked to the 5'end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
  • the regulatory sequence may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino acid terminus of the polypeptide and directs the encoded polypeptide into the cell's secretory pathway.
  • the 5'end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked to a translation reading frame having a segment encoding a secreted polypeptide.
  • the 5'end of the coding sequence may contain a signal peptide coding region foreign to the coding region. When the coding sequence does not naturally contain a signal peptide coding region, a foreign signal peptide coding region may be required.
  • the foreign signal peptide coding region can simply replace the natural signal peptide coding region to enhance secretion of the polypeptide.
  • any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell, that is, secreted into the culture medium, can be used in the present invention.
  • the nucleic acid construct of the invention is an expression cassette.
  • expression box refers to the complete elements required to express a gene, including the promoter, gene coding sequence, and PolyA plus tail signal sequence.
  • the invention also relates to vectors containing the polynucleotide or nucleic acid constructs of the invention, including expression vectors and cloning vectors.
  • the expression vector may be any vector (such as a plasmid or virus) that can conveniently undergo recombinant DNA methods and can result in the expression of the nucleotide sequence of interest.
  • Cloning vectors are usually able to multiply in host cells after being introduced into host cells.
  • the choice of vector generally depends on the compatibility of the vector with the host cell into which it is introduced.
  • the vector may be a linear or closed circular plasmid.
  • the vector may be a self-replicating vector, that is, exists as an extrachromosomal entity, and its replication does not depend on a vector for chromosomal replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome.
  • the carrier may contain any means for ensuring self-replication.
  • the vector may be a vector that, when introduced into a host cell, integrates into the genome and replicates with the chromosome into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon can be used.
  • the vector of the present invention preferably contains one or more selectable markers that allow easy selection of cells for transformation, transfection, transduction, etc.
  • Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophy, etc.
  • the vector of the present invention preferably contains elements that allow the vector to integrate into the genome of the host cell or that the vector replicates independently of the genome in the cell.
  • More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the yield of the gene product.
  • the increase in polynucleotide copy number can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene and the polynucleotide, which contains the amplified copy of the selectable marker gene and Such cells containing additional copies of polynucleotides can be selected by culturing the cells in the presence of a suitable selection agent.
  • the vector of the present invention preferably contains a synthetic sequence containing multiple restriction enzyme recognition sites, which can provide various insertable positions or insertion schemes for foreign DNA.
  • the expression vector of the present invention is more preferably a vector that can be used for expression in Pichia pastoris.
  • the vector of the present invention is preferably a vector used in commercial Pichia pastoris such as a series of vectors such as pPIC, pPICZ, pAO, pGAP, or pGAPZ.
  • the present invention also relates to recombinant host cells containing polynucleotide or nucleic acid constructs of the present invention used for recombinant production of polypeptides.
  • the vector including the polynucleotide of the present invention is introduced into the host cell so that the vector is maintained as a component of a chromosome as described earlier or as an extrachromosomal self-replicating vector.
  • the choice of host cell depends largely on the gene encoding the polypeptide and its source.
  • the host cell may be a unicellular microorganism or a non-unicellular microorganism.
  • Unicellular microorganisms such as Gram-positive bacteria, including, but not limited to, Bacillus cells, such as Bacillus alkaliphilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus stearothermophilus and Bacillus thuringiensis; etc.; or Streptomyces cells, such as Streptomyces viridans; or Gram-negative bacteria, such as Escherichia coli and Pseudomonas.
  • the bacterial hosts are Bacillus subtilis, E. coli, Bacillus licheniformis, Bacillus stearothermophilus and E. coli cells.
  • the host cell may also be a eukaryote, such as mammalian, insect, plant, yeast or fungal cells.
  • the host cell is a eukaryotic cell, as used herein, "eukaryotic" includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota etc.
  • the host cell is a cell of the Ascomycota genus such as Saccharomyces, Pichia, Yarrowia, Candida, Komagataella and the like.
  • the host cell is Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, etc. In another most preferred aspect, the host cell is a Pichia pastoris cell.
  • the polypeptide of the present invention can be produced by the following method, which includes: (a) cultivating a host cell containing an expression vector expressing the polypeptide under conditions conducive to the production of the polypeptide; and (b) recovery The polypeptide.
  • cells can be cultured in a medium suitable for producing polypeptides using methods known in the art.
  • cells can be cultured in shake flasks and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermentation tank, in a suitable medium and
  • the polypeptide is cultured under conditions that allow expression and/or isolation of the polypeptide.
  • the cultivation takes place in a suitable medium using methods known in the art including carbon and nitrogen sources and inorganic salts.
  • Suitable media can be obtained from commercial suppliers or can be prepared according to the disclosed compositions. If the polypeptide is secreted into the medium, the polypeptide can be directly recovered from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.
  • Peptide chemical synthesis methods include solid phase synthesis and liquid phase synthesis, of which solid phase synthesis is commonly used.
  • Solid-phase synthesis methods include but are not limited to Fmoc and tBoc.
  • resin is used as an insoluble solid phase carrier.
  • amino acids are connected to the peptide chain one by one from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus).
  • Each amino acid linking cycle consists of the following three-step reaction: 1) Protection: The protected amino acid must use a deprotection solvent to remove the protecting group of the amino group; 2) Activation: the carboxyl group of the amino acid to be linked is activated by the activator; and 3) Coupling: the activated carboxyl group is exposed to the previous amino acid The amino group reacts to form a peptide bond. The cycle is repeated until the peptide chain reaches the desired length. Finally, the connection between the peptide chain and the solid phase carrier is cleaved with a cutting solution, and the desired amino acid sequence can be obtained.
  • the above chemical synthesis can be carried out on a program-controlled automated peptide synthesizer. Such instruments include, but are not limited to, Tribute dual-channel peptide synthesizer launched by Protein Technologies, UV Biosystem’s UV Online Monitor system, and Aapptec’s Focus XC. Three-channel synthesizer, etc.
  • polypeptides described in the present invention can be recovered using methods known in the art.
  • the polypeptide can be recovered from the culture medium by conventional methods including, but not limited to, centrifugation, filtration, ultrafiltration, extraction, chromatography, spray drying, freeze drying, evaporation, or precipitation.
  • polypeptide of the present invention can be purified by various methods known in the art, including but not limited to chromatography (eg ion exchange, affinity, hydrophobicity, chromatographic focusing, molecular exclusion), electrophoresis (eg isoelectric focusing) ), differential solubility (such as salting out precipitation), SDS-PAGE or extraction methods to obtain substantially pure polypeptide.
  • chromatography eg ion exchange, affinity, hydrophobicity, chromatographic focusing, molecular exclusion
  • electrophoresis eg isoelectric focusing
  • differential solubility such as salting out precipitation
  • SDS-PAGE SDS-PAGE or extraction methods to obtain substantially pure polypeptide.
  • the polypeptide of the present invention has phospholipase C activity and has increased specific enzyme activity.
  • the polypeptide of the present invention can be applied to many aspects such as oil refining, phospholipid modification, feed improver, food industry and pharmaceutical industry, including but not limited to baking, detergent, improving the filterability of aqueous solution or syrup, etc. .
  • the polypeptide of the present invention can improve the degumming efficiency and increase the yield of glycolide (DAG) in the degumming process.
  • DAG glycolide
  • the polypeptide of the present invention may be provided in the form of a pure enzyme preparation or in the form of a composition.
  • the composition may be a powder composition, a liquid composition, or a paste composition.
  • the composition may contain different excipients depending on the different uses of the enzyme-containing composition. Excipients known in the art can be added to the composition of the present invention, such adjuncts include but are not limited to sorbitol, potassium sorbate, methyl benzoate, ethyl benzoate, sucrose, mannose, trehalose, starch , One or more of sodium chloride, calcium chloride and other stabilizers or other substances.
  • the amount of the polypeptide of the present invention used in the method of the present invention can be determined according to actual conditions.
  • the present invention also provides an enzymatic degumming method, which includes the step of adding the polypeptide of the present invention to crude oil for degumming.
  • the crude oil may be heated to 50 to 70°C, preferably 50 to 60°C, and then the polypeptide of the present invention, namely phospholipase C, may be added.
  • the phospholipase C of the present invention is usually added in the form of an aqueous solution. Based on the weight of crude oil, the amount of enzyme added is 10 to 1000 ppm, preferably 50 to 500 ppm, and more preferably 100 to 300 ppm.
  • Degumming conditions generally include: stirring at 50 to 60°C for 1 to 3 hours, and then heating to 80 to 90°C for 1 to 10 minutes.
  • Crude oils suitable for degumming in the method of the present invention include, but are not limited to, soybean oil, sunflower oil, peanut oil, rapeseed oil, rice bran oil, corn oil, olive oil, palm oil, palm kernel oil, palm soft butter, canola oil , Castor oil, coconut oil, coriander oil, cottonseed oil, hazelnut oil, hemp seed oil, flaxseed oil, mango kernel oil, white mango flower oil, cattle hoof oil, safflower oil, camellia oil, tall oil, chun oil and Other vegetable oils.
  • Pichia pastoris SMD1168 Invitrogen, Catalog No. C17500
  • E. coli DH5a TAKARA: Catalog#.D9057A
  • LB liquid medium 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0.
  • LB solid medium Add 1.5% agar to LB liquid medium.
  • YPD liquid medium 1% yeast extract, 2% peptone, 2% glucose.
  • YPD solid medium Add 2% agar to LB liquid medium.
  • MGYS solid medium 1.34% yeast nitrogen source base (YNB) containing ammonium sulfate without amino acids, 1% glycerol, 1M sorbitol, 4 ⁇ 10 -5 % D-biotin, 2% agar.
  • yeast nitrogen source base YNB
  • BMM-soybean phospholipid screening medium 1.34% yeast nitrogen source base (YNB) containing ammonium sulfate without amino acids, 4 ⁇ 10 -5 % D-biotin, 0.5% methanol (added after sterilization), 2% soybean phospholipid emulsification Solution, 0.1M citric acid-sodium citrate buffer (pH 6.6), 2% agar, 10uM ZnSO 4 ⁇ 7H 2 O was added.
  • yeast nitrogen source base YNB
  • ammonium sulfate without amino acids
  • 4 ⁇ 10 -5 % D-biotin 0.5% methanol (added after sterilization)
  • soybean phospholipid emulsification Solution 0.1M citric acid-sodium citrate buffer (pH 6.6)
  • 2% agar 10uM ZnSO 4 ⁇ 7H 2 O was added.
  • soybean phospholipid emulsion 2 g soybean phospholipid, 100 ml H 2 O, homogenized with a high-speed homogenizer at 8000 rpm for 1 min.
  • BMGY liquid medium 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) containing ammonium sulfate without amino acids, 1% glycerol, 4 ⁇ 10 -5 % D-biotin, 0.1M diphosphate Potassium hydrogen-dipotassium phosphate buffer (pH 6.0).
  • yeast nitrogen source base YNB
  • BMMY liquid medium 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) containing ammonium sulfate without amino acids, 0.3% ZnSO 4 ⁇ 7H 2 O, 0.5% methanol (added after sterilization), 4 ⁇ 10 -5 % D-Biotin (added after sterilization), 0.1 M citric acid-sodium citrate buffer (pH 6.6).
  • yeast nitrogen source base YNB
  • Sample enzyme activity (U/ml) A (410nm absorbance) x 0.1935 x dilution factor x 10/15.
  • the improved Bradford method protein concentration determination kit was purchased from Shanghai Shenggong Biological Engineering Co., Ltd.; the PCR enzyme was DNA Polymerase was purchased from Bao Bioengineering (Dalian) Co., Ltd.; T4 DNA ligase was purchased from Fuzyme Tais Co., Ltd.
  • the plasmid was extracted, linearized with SalI, and a fragment of about 8.5 kb was recovered. Take 500ng vector, and transform the vector into competent cells of Pichia pastoris SMD1168 strain by electric transformation method. The transformants were inoculated on MGYS plates and cultured at 30°C for 3 days to obtain a Pichia pastoris mutant library of PLC-N63DN131SN134D-Y56H. Pick the monoclonal on the plate and go to the BMM-soybean phospholipid screening plate. Select one of the clones with a relatively large white precipitation circle, number 31#.
  • Strain 31# was inoculated in 3ml YPD liquid medium and cultured at 30°C overnight to extract genomic DNA. Using the genomic DNA of strain 31# as a template, use DNA polymerase and primers were used for PCR amplification of AOX1-5/AOX1-3 to obtain the DNA sequence of PLC in strain 31#. Send the obtained sequence to Shanghai Biotech Engineering Co., Ltd. to sequence AOX1-5/AOX1-3 with primers:
  • AOX1-5 CGACTGGTTCCAATTGACAACG (SEQ ID NO: 5);
  • AOX1-3 GGCAAATGGCATTCTGACATCCTC (SEQ ID NO: 6).
  • the DNA sequencing result of PLC 31# is described in SEQ ID NO:3.
  • the DNA sequence of PLC 31# has 5 bases mutated, and the sequence encoding the amino acid is described in SEQ ID NO:4
  • the amino acids at positions 6, 8, 10, and 104 of the amino acid sequence were mutated from lysine, lysine, glycine, and lysine to proline, valine, serine, and serine, respectively.
  • induction was carried out with 2% methanol, 1% methanol was added after 24h and 32h, 1% methanol was added after 48h and 56h, and samples were taken at 72h.
  • the obtained sample was subjected to ultrafiltration desalting and concentration by a 40-fold ultrafiltration tube with a molecular weight cutoff of 10 kDa.
  • the treated sample was added to the buffer (20 mM citric acid-sodium citrate buffer (pH 6.6), 10 uM ZnSO 4 ).

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Abstract

本发明提供一种高酶活的磷脂酶C突变体，其序列如SEQ ID NO:7所示的多肽，或在SEQ ID NO:7所述的多肽中经过取代、缺失或添加一个或几个氨基酸，同时保留SEQ ID NO：7所具备的磷脂酶C活性的由其衍生的多肽。本发明的磷脂酶C突变体能提高脱胶效率，增加脱胶过程中甘二酯(DAG)的得率。

Description

一种高酶活的磷脂酶C突变体 技术领域
本发明涉及一种高酶活的磷脂酶C突变体。
背景技术
脱胶是油脂精炼的一个重要步骤，而传统的水化脱胶法经济成本高，物料能耗大，环境污染重，所以近些年，很多努力致力于将酶法脱胶用于油脂精炼中的脱胶环节，并取得了很大的进展。同传统方法相比,酶法脱胶能够提高经济效益，做到节能减排，对生态环境污染少，在环保、经济、质量等方面具有较大的优势。油脂脱胶所用的酶即为磷脂酶，并且同其他脱胶酶相比，磷脂酶C(PLC)表现出更大的优势，比如，增加甘二酯(DAG)的得率，以及减少得油量的损失。
Bacillus cereus的PC-PLC(BC-PC-PLC)是研究较早的一种磷脂酶C。BC-PC-PLC全长为283个氨基酸，其中包含24个氨基酸的信号肽和14个氨基酸的前导肽，成熟肽为245个氨基酸(Johansen et al.1988)。BC-PC-PLC的晶体结构已经被报道，其由多个螺旋结构域组成，催化位点为55位天冬氨酸，并且含有至少三个Zn2+结合位点(Hough et al.1989)。BC-PC-PLC的异源表达研究较少目前只在Bacillus subtilis和pichia pastoris中进行表达(Durban et al.2007；Seo et al.2004)。
在之前的研究中，发明人已经通过将BC-PC-PLC的四个氨基酸即56位，63位，131位和134位天冬酰胺分别突变为组氨酸，天冬氨酸，丝氨酸和天冬氨酸，比酶活相比于野生型大大提高。为了进一步提高该磷脂酶C的比酶活力和脱胶效率，发明人选择了BC-PC-PLC蛋白中第6位，第8位，第10位，第104位，第205位氨基酸进行随机饱和突变，对已有的磷脂酶C突变体进行定向进化，希望能够得到比酶活进一步提高的突变体，从而能够得到更高效的磷脂酶C，提高脱胶效率，增加脱胶过程中甘二酯(DAG)的得率。
发明内容
本发明提供一种分离的多肽，所述多肽选自：
(1)SEQ ID NO:7所示的多肽；和
(2)与SEQ ID NO:7至少80％，优选至少85％，更优选至少90％，更优选至少95％，更优选至少97％，更优选至少98％，更优选至少99％的序列同一性，对应于SEQ ID NO:7第6位、第8位、第10位和第104位中的至少1个位置的氨基酸残基分别相应地与SEQ ID NO:7第6位、第8位、第10位和/或第104位相同，同时保留SEQ ID NO：7所具备的磷脂酶C活性的多肽。
在一个或多个实施方案中，SEQ ID NO:7中，第6位氨基酸残基是脯氨酸或色氨酸；第8位氨基酸残基是丙氨酸、亮氨酸或异亮氨酸；第10位和第104位氨基酸各自独立为丝氨酸或苏氨酸。
在一个或多个实施方案中，所述分离的多肽如SEQ ID NO:4所示。
本发明还提供一种分离的多肽，所述多肽与SEQ ID NO:4所示的氨基酸序列具有至少80％，优选至少85％，更优选至少90％，更优选至少95％，更优选至少97％，更优选至少98％，更优选至少99％的序列同一性，且所述分离多肽在对应于SEQ ID NO:4第6、8、10和104位上的氨基酸残基分别是脯氨酸、缬氨酸、丝氨酸和丝氨酸；优选的，所述多肽获自枯草芽孢杆菌(Bacillus subtilis)。
本发明还提供一种多核苷酸序列，选自：
(1)编码本文任一实施方案所述多肽的多核苷酸序列；和
(2)(1)所述多核苷酸序列的互补序列；和
(3)(1)或(2)所述序列的长15-30个碱基的片段；
优选地，所述多核苷酸序列如SEQ ID NO:3所示。
本发明还提供一种核酸构建体，该核酸构建体包含本文任一实施方案所述的多核苷酸序列和一条或多条与该多核苷酸序列操作性连接的调控序列。
在一个或多个实施方案中，所述核酸构建体为载体。
在一个或多个实施方案中，所述载体为表达载体或克隆载体。
本发明还提供一种遗传工程化的宿主细胞，所述宿主细胞含有本文任一实施方案所述的多核苷酸序列或核酸构建体。
本发明还提供一种组合物，所述组合物含有本文任一实施方案所述的多肽和任选的辅料，优选地，所述辅料为选自活性炭、氧化铝、硅藻土、多孔陶瓷、多孔玻璃的吸附材料。
本发明还提供本文任一实施方案所述的多肽、多核苷酸序列、核酸构建体、宿主细胞或组合物在油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业中的应用。
本发明还提供一种酶法脱胶方法，所述方法包括使用本文任一实施方案所述的多肽进行脱胶，优选的，所述方法包括将所述多肽与毛油接触的步骤，优选的，所述步骤包括将所述多肽与50-70℃毛油接触。
在一个或多个实施方案中，所述方法包括以下特征中的一项或多项：
(1)以毛油重量计，所述多肽的添加量为10～1000ppm，优选为50～500ppm，更优选为100～300ppm；
(2)所述脱胶包括：在50～60℃下搅拌1～3小时，然后升温至80～90℃保持1～10分钟；和
(3)所述毛油选自：大豆油、葵花籽油、花生油、菜籽油、米糠油、玉米油、橄榄油、棕榈油、棕榈仁油、棕榈软脂、卡诺拉油、蓖麻油、椰子油、芫荽油、棉籽油、榛子油、***籽油、亚麻籽油、芒果仁油、白芒花油、牛蹄油、红花油、山茶花油、妥尔油和椿油。
附图说明
图1：突变体的比酶活。
图2：脱胶小试结果。
图3：磷脂酶活力测定标准曲线。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本发明提供氨基酸序列如SEQ ID NO：7所示的分离的多肽，或与SEQ ID NO:7至少80％，优选至少85％，更优选至少90％，更优选至少95％，更优选至 少97％，更优选至少98％，更优选至少99％的序列同一性，且对应于SEQ ID NO:7第6位、第8位、第10位和第104位中的至少1个位置的氨基酸残基分别相应地与SEQ ID NO:7第6位、第8位、第10位和/或第104位相同，同时保留SEQ ID NO：7所具备的磷脂酶C活性的多肽。
本文中，“分离的”意指在自然界中不存在的一种形式或物质。分离的物质的非限制性例子包括任何非天然存在的物质和至少部分地从与其在自然界中相关联的一种或多种或全部天然存在的组分中除去的任何物质，包括但不限于任何酶、变体、核酸、蛋白质、肽或辅因子。SEQ ID NO:7中，第6位和第8位氨基酸残基(Xaa)可以是具有非极性侧链的氨基酸残基，包括但不限于丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸；第10位和第104位氨基酸残基(Xaa)可以是具有不带电荷的极性侧链的氨基酸，包括但不限于甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸。优选地，SEQ ID NO:7中，第6位氨基酸残基是脯氨酸或色氨酸；第8位氨基酸残基是丙氨酸、亮氨酸或异亮氨酸；第10位和第104位氨基酸各自独立为丝氨酸或苏氨酸。示例性的多肽的氨基酸序列如SEQ ID NO:4所示。
本发明还包括在SEQ ID NO：7的基础上具有一个或多个(通常为1-10个，例如1、2、3、4、5、6、7、8、9或10个)氨基酸突变(缺失、***和/或取代)、同时保留了SEQ ID NO:4所示氨基酸序列的磷脂酶C活性的多肽。在某些实施方案中，所述氨基酸突变是在SEQ ID NO:7的C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为8以内)氨基酸的多肽。应理解的是，所述的一个或多个氨基酸突变通常不包括SEQ ID NO:7第6、8、10和104位上的氨基酸(Xaa)全部发生突变的情况。
突变优选为保守性取代。例如，在本领域中，用性能相近或相似的氨基酸进行保守性取代时，通常不会改变蛋白质或多肽的功能。“性能相近或相似的氨基酸”包括例如，具有相似侧链的氨基酸残基的家族，这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、 具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，在本发明多肽中用来自同一侧链类的另一氨基酸残基替换一个或几个位点，将不会在实质上影响其活性。
在本发明中，术语“随机饱和突变”指将需要突变的位点在PCR引物中以设计为NNK兼并密码子，从而能够覆盖所有20种氨基酸达到饱和突变的效果，又由于选择了多个位点进行突变，其组合方式具有随机性，因此作者将此方法称之为随机饱和突变。
此外，本领域技术人员公知，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的蛋白末端引入了一个或多个不相干的残基，而这并不影响目的蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸序列添加至重组蛋白的N末端、C末端或该蛋白内的其它合适区域内，这类氨基酸序列包括但不限于接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、标签(如6His或Flag)、或合适的蛋白水解酶位点等。应理解，这些氨基酸序列的存在不会影响到所得多肽的活性。因此，本发明也包括在本发明多肽的C末端和/或N末端或其蛋白内合适的区域内具有一个或数个利于该多肽表达载体的构建、该多肽的表达和/或纯化的氨基酸的多肽，这些多肽仍具有本文所述的磷脂酶C活性。
因此，在某些实施方案中，本发明包括与SEQ ID NO:7具有至少80％、优选至少85％、更优选至少90％、更优选至少95％、更优选至少96％、更优选至少97％、更优选至少98％、更优选至少99％的序列同一性的氨基酸序列。更进一步地，本发明包括与SEQ ID NO:4具有至少80％、优选至少85％、更优选至少90％、更优选至少95％、更优选至少96％、更优选至少97％、更优选至少98％、更优选至少99％的序列同一性的氨基酸序列；且与SEQ ID NO:4和7具有所述序列同一性的氨基酸序列在对应于SEQ ID NO:4和7第6、8、10和104位上的氨基酸残基未全部发生突变，优选为仍保持为SEQ ID NO:4和7相应位置上的氨基酸残基，例如仍保持为SEQ ID NO:4第6、8、10和104位上的脯氨酸、缬氨酸、丝氨酸和丝氨酸。更优选地，具有所述序列同一性的氨基酸序列获自枯 草芽孢杆菌(Bacillus subtilis)。本文中，序列相同性用于描述两个氨基酸序列之间或两个核苷酸序列之间的相关性。可使用本领域周知的方法来计算序列相同性。例如，可使用EMBOSS包(EMBOSS：欧洲分子生物学开放软件套件，赖斯等，2000，遗传学趋势，16:276-277)的尼德尔(Needle)程序中所实施的尼德尔曼-翁施(Needleman-Wunsch)算法(Needleman和Wunsch，1970，分子生物学杂志，48:443-453)来确定两个氨基酸序列之间的序列相同性。或者，可使用NCBI上的BLASTP来计算两条氨基酸序列之间的序列相同性。
根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。
多核苷酸
本申请包括编码本发明多肽的核苷酸序列或其互补序列。SEQ ID NO：3显示了本发明多肽的编码序列例子。所述“编码序列”包括编码本发明所述多肽(尤其是SEQ ID NO：7)的核酸序列。编码本发明多肽的序列可以与例如SEQ ID NO：3所示的编码区序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指相同的氨基酸序列，但核苷酸序列有差别的核苷酸序列。
编码本发明多肽的序列包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的片段、类似物、衍生物和变异形式。这些核苷酸变异体包括取代变异体、缺失变异体和***变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或***，但不会从实质上改变其编码的蛋白的功能。
本发明也包括编码本发明多肽的核酸序列(如SEQ ID NO：3或其互补序列)的片段。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)以确定和/或分离编码本发明多肽的多核苷酸。因此，在某些实施例中，核酸片段的长度在15-30个碱基。可采用现有技术从本 发明的核酸序列中挑选出适当的核酸片段，用作引物或探针。
本发明的多肽的编码序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
核酸构建体
本发明也涉及包括与指导编码序列在合适宿主细胞中，在与该调控序列相适合的条件下表达的一个或多个调控序列可操作连接的本发明的分离多核苷酸的核酸构建体。术语“可操作连接”指调控序列位于适当的位置从而能控制、指导感兴趣的多核苷酸序列的表达。编码本发明多肽的多核苷酸可以多种方式***作以保证该多肽的表达。
调控序列可以是合适的启动子序列，为用于表达编码本发明多肽的多核苷酸的宿主细胞识别的核苷酸序列。启动子序列包含接到多肽表达的转录调控序列。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
用于指导本发明的核酸构建体，特别是在细菌宿主细胞中转录的合适启动子的实例是从噬菌体T7启动子、大肠杆菌lac操纵子、天蓝色链霉菌(Streptomyces coelicolor)琼脂糖酶基因、枯草芽孢杆菌果聚糖蔗糖酶基因、地衣芽孢杆菌α-淀粉酶基因、解淀粉芽孢杆菌α-淀粉酶基因、地衣芽孢杆菌青霉素酶基因等中获得的启动子序列。
用于指导本发明的核酸构建体在丝状真菌宿主细胞正转录的合适启动子的实例是从米曲霉TAKA淀粉酶、米黑根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定的α-淀粉酶、黑曲霉或泡盛曲霉糖化酶(glaA)、里氏木霉纤维二糖水解酶I、氏木霉纤维二糖水解酶II、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、里氏木霉葡聚糖内切酶等基因获得的启动子 及其突变、截短和杂合(hybrid)启动子。
在酵母宿主中，有用的启动子可获得自酿酒酵母烯醇酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母乙醇脱氢酶、3-磷酸甘油醛脱氢酶、酿酒酵母磷酸丙糖异构酶、酿酒酵母3-磷酸甘油酸激酶的基因、巴斯德毕赤酵母醇氧化酶基因。用于酵母宿主细胞的其它有用启动子由Romanos et al.，1992，Yeast 8:423-488描述。
调控序列也可以是合适的转录终止子序列，为宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3′末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
用于细菌宿主的优选终止子可以是来自T7噬菌体的终止子。
用于丝状真菌宿主细胞的优选终止子获得自米曲霉TAKA淀粉酶、黑曲霉葡萄糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉α-葡萄糖苷酶的基因。
用于酵母宿主细胞的优选终止子获得自酿酒酵母烯醇酶、酿酒酵母细胞色素C、酿酒酵母甘油醛-3-磷酸脱氢酶、巴斯德毕赤酵母醇氧化酶基因等。
调控序列也可以是合适的前导序列，对宿主细胞翻译重要的mRNA的非翻译区。签到序列与编码该多肽的核苷酸序列的5′末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
调控序列也可以是编码与多肽的氨基酸末端连接的氨基酸序列并且指导该编码的多肽进入细胞分泌途径的信号肽编码区。核苷酸序列编码序列的5′端可固有地包含天然连接具有编码分泌多肽的编码区节段的翻译阅读框的信号肽编码区。备选地，编码序列的5′端可包含与该编码区外源的信号肽编码区。当编码序列非天然地包含信号肽编码区时，可能需要外来的信号肽编码区。备选地，外来的信号肽编码区可简单地替换天然的信号肽编码区以增强多肽的分泌。然而，指导表达的多肽进入选择的宿主细胞的分泌途径的任何信号肽编码区，即分泌进入培养基，都可用于本发明。
在某些实施方案中，本发明的核酸构建体为表达框。术语“表达框”是指表达一个基因所需的完整元件，包括启动子、基因编码序列、PolyA加尾信号序列。
载体
本发明也涉及含有本发明多核苷酸或核酸构建体的载体，包括表达载体和克隆载体。表达载体可以是能够方便地经受重组DNA方法并且可导致感兴趣的核苷酸序列表达的任何载体(如质粒或病毒)。克隆载体通常在引入到宿主细胞中后能在宿主细胞中大量繁殖。
载体的选择一般取决于载体与其中被导入该载体的宿主细胞的相容性。该载体可以是线性或闭合的环形质粒。
载体可以是自主复制的载体，即作为染色体外实体存在，其复制不依赖于染色体复制的载体，例如质粒、染色体外元件、微型染色体或人工染色体。载体可包含用于保证自我复制的任何方式。备选地，载体可以是当被导入宿主细胞时，整合到基因组中并且与其已经被整合进入的染色体一起复制的载体。此外，可使用一起包含将被导入宿主细胞基因组的总DNA的单个载体或质粒或两个或多个载体或质粒，或转座子。
本发明的载体优选包含一个或多个容许容易选择转化、转染、转导等细胞的可选择标记。可选择的标记是基因，其产物提供对抗生素或病毒的抗性、对重金属的抗性、原养型至营养缺陷型等。
本发明的载体优选包含容许该载体整合进入宿主细胞基因组或该载体在细胞中独立于基因组自主个复制的元件。
一个以上拷贝的本发明的多核苷酸可被***宿主细胞以增加该基因产物的产量。多核苷酸拷贝数的增加可通过将至少一个附加拷贝的序列整合进入宿主细胞基因组或通过包括可扩增的选择标记基因和该多核苷酸来获得，其中包含扩增拷贝的选择标记基因并且由此包含附加拷贝多核苷酸的细胞可通过在存在适当的选择剂时培养该细胞来筛选。
本发明的载体优选包含人工合成的一段序列，含有多个限制内切酶识别位点，能为外源DNA提供多种可***的位置或***方案。
本发明的表达载体更为优选地选择可用于毕赤酵母中表达的载体。本发明的载体优选商品化的毕赤酵母中使用的载体如pPIC、pPICZ、pAO、pGAP或pGAPZ等一系列的载体。
宿主细胞
本发明也涉及包括被用于重组生产多肽的含有本发明多核苷酸或核酸构建体的重组宿主细胞。包括本发明多核苷酸的载体被导入宿主细胞以使得该载体如早先说明的作为染色体的组成部分或作为染色体外的自我复制载体来维持。宿主细胞的选择很大程度上取决于编码多肽的基因及其来源。
宿主细胞可以是单细胞微生物或非单细胞微生物。单细胞微生物例如***，包括但不限于芽孢杆菌细胞，例如，嗜碱芽孢杆菌、解淀粉芽孢杆菌、短芽孢杆菌、巨大芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌、凝结芽孢杆菌、嗜热脂肪芽孢杆菌和苏云金芽孢杆菌等；或链霉菌细胞，例如钱青紫链霉菌；或革兰氏阴性细菌，例如大肠杆菌和假单胞菌属。在优选的方面，细菌宿主是枯草芽孢杆菌、大肠杆菌、地衣芽孢杆菌、嗜热脂肪芽孢杆菌和大肠杆菌细胞。
宿主细胞也可以是真核生物，例如哺乳动物、昆虫、植物、酵母或真菌细胞。在优选的方面，宿主细胞是真核细胞，如在此使用的“真核”包括子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)、接合菌门(Zygomycota)以及卵菌门等。
在更优选的方面，宿主细胞是子囊菌门的细胞如酵母属(Saccharomyces)、毕赤酵母属(Pichia)、耶氏酵母属(Yarrowia)、假丝酵母属(Candida)以及Komagataella属等。
在最优选的方面，宿主细胞是巴斯德毕赤酵母(Pichia pastoris)、酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolytica)等。在另外最优选方面，宿主细胞是巴斯德毕赤酵母(Pichia pastoris)细胞。
生产方法
在获得多肽的编码序列后，可采用如下方法生产本发明多肽，该方法包括：(a)在有助于生产多肽的条件下培养含表达该多肽的表达载体的宿主细胞；以及(b)回收该多肽。
本发明的生产方法中，细胞可以利用本领域已知的方法在适于生产多肽的培养基中培养。例如，细胞可通过在实验室或工业发酵罐中进行的摇瓶培养和小规模或大规模的发酵(包括连续的、分批的、分批补料或固态发酵)，在合适的培养基和容许该多肽表达和/或分离的条件下进行培养。培养发生在利用本领域已知 的方法包括碳源和氮源和无机盐的合适培养基中。合适的培养基可获得自商业供应者或可根据公开的组合物来制备。如果该多肽分泌进入培养基，该多肽可从培养基直接回收。如果该多肽不分泌进入培养基，它可从细胞裂解物回收。
或者，也可采用本领域已知的化学合成方法来合成本发明的多肽。多肽化学合成方法包括固相合成法和液相合成法，其中以固相合成法常用。固相合成方法包括但不限于Fmoc和tBoc两种常用方法。通常，使用树脂作为不溶性的固相载体，通常从C端(羧基端)向N端(氨基端)逐个将氨基酸连接在肽链上，每个氨基酸连接循环由以下三步反应构成：1)脱保护：被保护的氨基酸必须用一种脱保护溶剂去除氨基的保护基团；2)活化：待连接的氨基酸的羧基被活化剂所活化；和3)偶联：活化的羧基与前一个氨基酸裸露的氨基反应，形成肽键。反复循环直到肽链延伸至所需长度时即可完成。最后用切割液切割肽链和固相载体之间的连接，就可获得所需的氨基酸序列。可以在程序控制的自动化多肽合成仪上进行上述化学合成，这类仪器包括但不限于Protein Technologies公司推出的Tribute双通道多肽合成仪、C S Bio公司的UV Online Monitor***、Aapptec公司推出的Focus XC三通道合成仪等。
本发明所描述的多肽可利用本领域已知的方法来回收。例如，多肽可通过常规方法，包括但不限于离心、过滤、超滤、萃取、层析、喷雾干燥、冷冻干燥、蒸发或沉淀等从培养基回收。
本发明的多肽可通过本领域已知的多种方法来纯化，包括但不限于色谱法(如离子交换、亲和性、疏水性、色谱聚焦、分子排阻)，电泳法(例如等电聚焦)、差异溶解度(如盐析沉淀)、SDS-PAGE或萃取法以获得基本上纯的多肽。
多肽的性能及用途
本发明的多肽具有磷脂酶C活性，具有提高的比酶活。本发明的多肽可应用于油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业等多个方面，包括但不限于用于烘烤、用于洗涤剂、改善水溶液或糖浆的过滤性等。在用于脱胶时，本发明的多肽能提高脱胶效率，增加脱胶过程中甘二酯(DAG)的得率。
本发明的多肽可以纯的酶制品的形式提供，也可以组合物的形式提供。组合物可以是粉末组合物，也可以是液体组合物，或糊状组合物。以组合物形式提供 时，根据该含酶组合物的不同用途，该组合物可含有不同的辅料。可将本领域已知的辅料添加到本发明的组合物中，这类辅料包括但不限于山梨醇、山梨酸钾、苯甲酸甲酯、苯甲酸乙酯、蔗糖、甘露糖、海藻糖、淀粉、氯化钠、氯化钙等稳定剂或其他物质中的一种或多种。
本发明方法中使用的本发明多肽的量可根据实际情况加以确定。
酶法脱胶
本发明还提供一种酶法脱胶方法，所述方法包括将本发明的多肽加到毛油中进行脱胶的步骤。具体而言，可先将毛油加热至50～70℃，优选50～60℃，然后再加入本发明的多肽，即磷脂酶C。
本发明的磷脂酶C通常以水溶液的形式加入。以毛油重量计，酶的添加量为10～1000ppm，优选为50～500ppm，更优选为100～300ppm。
脱胶条件一般包括：在50～60℃下搅拌1～3小时，然后升温至80～90℃保持1～10分钟。
适用于本发明方法脱胶的毛油包括但不限于大豆油、葵花籽油、花生油、菜籽油、米糠油、玉米油、橄榄油、棕榈油、棕榈仁油、棕榈软脂、卡诺拉油、蓖麻油、椰子油、芫荽油、棉籽油、榛子油、***籽油、亚麻籽油、芒果仁油、白芒花油、牛蹄油、红花油、山茶花油、妥尔油、椿油和其他植物油。
下文将以具体实施例的方式阐述本发明。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等，《分子克隆：实验室指南》(美国纽约州：冷泉港实验室出版社(Cold Spring Harbor Laboratory Press)，1989)所述的条件，或按照制造厂商所建议的条件进行。对于试剂的用法和用量，除非另有说明，否则按照常规的用法和用量使用。
实验材料
1、实验菌株和质粒
菌株：毕赤酵母SMD1168(Invitrogen，货号C17500)，大肠杆菌DH5a(TAKARA:Catalog#.D9057A)。
2、培养基和溶液
LB液体培养基：0.5％酵母提取物，1％胰化蛋白胨，1％NaCl，pH7.0。
LB固体培养基：在LB液体培养基中加入琼脂浓度1.5％。
YPD液体培养基：1％酵母提取物，2％蛋白胨，2％葡萄糖。
YPD固体培养基：在LB液体培养基中加入琼脂浓度2％。
MGYS固体培养基：1.34％酵母氮源碱(YNB)含硫酸铵不含氨基酸，1％甘油，1M山梨醇，4×10 -5％D-生物素，2％琼脂。
BMM-大豆磷脂筛选培养基：1.34％酵母氮源碱(YNB)含硫酸铵不含氨基酸，4×10 -5％D-生物素，0.5％甲醇(灭菌后加入)，2％大豆磷脂乳化液，0.1M柠檬酸-柠檬酸钠缓冲液(pH6.6)，2％琼脂，添加10uM的ZnSO 4·7H 2O。
2％大豆磷脂乳化液：2g大豆磷脂，100ml H 2O，用高速匀浆机8000rpm匀浆1min。
BMGY液体培养基：1％酵母提取物，2％蛋白胨，1.34％酵母氮源碱(YNB)含硫酸铵不含氨基酸，1％甘油，4×10 -5％D-生物素，0.1M磷酸二氢钾-磷酸氢二钾缓冲液(pH6.0)。
BMMY液体培养基：1％酵母提取物，2％蛋白胨，1.34％酵母氮源碱(YNB)含硫酸铵不含氨基酸，0.3％ZnSO 4·7H 2O，0.5％甲醇(灭菌后加入)，4×10 -5％D-生物素(灭菌后加入)，0.1M柠檬酸-柠檬酸钠缓冲液(pH6.6)。
3、酶活测定方法pNPPC法
磷脂酶活力测定标准曲线的绘制：
称取0.01391g对硝基苯酚，溶于50ml无菌水中配成2mmol/L工作液。各种试剂的加量见表制作标准曲线时的反应体积和反应条件与试验中测定样品酶活的条件相一致。
编号 1 2 3 4 5 6 7
2.0mmol/L pNP(ul) 0 7.5 15 22.5 30 37.5 45
ddH 2O(ul) 62.5 55 47.5 40 32.5 25 17.5
底物缓冲液(ml) 562.5 562.5 562.5 562.5 562.5 562.5 562.5
pNP总量(umol) 0 0.06 0.12 0.18 0.24 0.30 0.36
以上各种溶液混合后，在37℃处理15分钟，加500ul 0.5N的NaOH，410nm测定吸光度。所得标准曲线如图3所示。
4、反应缓冲液
0.1M硼酸-硼酸钠缓冲液(pH 7.6)，20mM pNPPC。
5、酶活计算
取600ul上述反应缓冲液，加入25ul待测酶液，37℃反应15min，加500ul 0.5N的NaOH终止反应，410nm测定吸光度。
样品酶活(U/ml)＝A(410nm吸光度)×0.1935×稀释倍数×10/15。
改良型Bradford法蛋白浓度测定试剂盒购自上海生工生物工程有限公司；PCR酶为
Figure PCTCN2019128970-appb-000001
DNA Polymerase，购自宝生物工程(大连)有限公司；T4 DNA连接酶购自富酶泰斯有限公司。
实施例1：饱和突变体文库构建及筛选
以pmAO-PLC-N63DN131SN134D-Y56H载体(见CN 201680072289.5，PLC-N63DN131SN134D-Y56H的DNA序列如SEQ ID NO:1所示，其编码的氨基酸序列如SEQ ID NO:2所示)为模板，委托苏州泓迅生物科技股份有限公司构建第6位，第8位，第10位，第104位，第205位氨基酸进行随机饱和突变文库。将质粒文库转化入大肠杆菌DH5α菌株，将所有大肠杆菌克隆洗至LB液体培养基中(含100μg/ml氨苄青霉素)，37℃培养4h。抽提质粒，用SalI进行线性化，回收约8.5kb的片段。取500ng载体，用电转化法将载体转化至毕赤酵母SMD1168菌株的感受态细胞中。将转化物接种于MGYS平板上，30℃培养3天，得到PLC-N63DN131SN134D-Y56H的毕赤酵母突变体文库。挑取平板上的单克隆，至BMM-大豆磷脂筛选平板上。选取白色沉淀圈相对较大的克隆之一，编号 31#。
实施例2：31#突变体序列分析
将31#菌株接种于3ml YPD液体培养基中，30℃培养过夜，抽提基因组DNA。以31#菌株的基因组DNA为模板，使用
Figure PCTCN2019128970-appb-000002
DNA聚合酶和引物对AOX1-5/AOX1-3进行PCR扩增，得到31#菌株中PLC的DNA序列。将得到的序列送往上海生工生物工程公司，用引物对AOX1-5/AOX1-3进行测序：
AOX1-5：CGACTGGTTCCAATTGACAACG(SEQ ID NO:5)；
AOX1-3：GGCAAATGGCATTCTGACATCCTC(SEQ ID NO:6)。
31#的PLC的DNA测序结果如SEQ ID NO:3所述，根据测序结果，31#的PLC的DNA序列有5个碱基发生了突变，其编码氨基酸的序列如SEQ ID NO:4所述，其氨基酸序列的第6位、第8位、第10位和第104位氨基酸分别由赖氨酸、赖氨酸、甘氨酸和赖氨酸突变为脯氨酸、缬氨酸、丝氨酸和丝氨酸。
实施例3：31#突变体摇瓶发酵酶活
取31#菌株及母本菌株(即转入了pmAO-PLC-N63DN131SN134D-Y56H载体的SMD1168)，先在液体YPD中活化，然后接种于BMGY培养基中，在30℃下，220rpm振荡培养过夜。将培养物转至BMMY培养基中，初始OD600为6。
首先，用2％甲醇进行诱导，在24h和32h后各补加1％甲醇，48h和56h后各补加1％甲醇，72h取样。将获得的样品用截留分子量为10kDa的超滤管进行超滤脱盐浓缩40倍。将处理后的样品加入缓冲液中(20mM柠檬酸-柠檬酸钠缓冲液(pH6.6)，10uM ZnSO 4)。
取0.5μl发酵液浓缩液至600ul pNPPC反应缓冲液中，37℃反应15min，加500ul 0.5N的NaOH终止反应，410nm测定吸光度。根据标准曲线，计算得到各个发酵液样品的PLC活力。用Bradford试剂测定摇瓶发酵液的蛋白浓度，从而得出比酶活。结果如图1所示，31#突变体发酵液样品的比酶活较母本菌株发酵液样品的比酶活有1.88倍的提高。
实施例4：31#脱胶小试
取大豆毛油100g，加热至55℃，分别加入50ppm和100ppm实施例3获得的31#突变体发酵液样品和母本菌株发酵液样品，使得体系中的水相为3％，用高速剪切机高速剪切(10000r/min)1min，在55℃下搅拌(750r/min)反应2h，升温至85℃，维持5min，样品12000rpm离心10min，取约10g上层油样，用HPLC检测其DAG含量。31#突变体发酵液样品和母本菌株发酵液样品相对于毛油的DAG增量如图2所示，31#突变体发酵液样品50ppm酶用量时的DAG增量与母本菌株发酵液样品100ppm时的DAG增量相同。因此31#突变体发酵液样品脱胶时的酶用量可以较母本菌株发酵液样品减少1倍。

Claims (10)

  1. 一种分离的多肽，其特征在于，所述多肽选自：
    (1)SEQ ID NO:7所示的多肽；和
    (2)与SEQ ID NO:7具有至少80％，优选至少85％，更优选至少90％，更优选至少95％，更优选至少97％，更优选至少98％，更优选至少99％的序列同一性，且对应于SEQ ID NO:7第6位、第8位、第10位和第104位中的至少1个位置的氨基酸残基分别相应地与SEQ ID NO:7第6位、第8位、第10位和/或第104位相同，同时保留SEQ ID NO：7所具备的磷脂酶C活性的多肽。
  2. 如权利要求1所述的分离的多肽，其特征在于，SEQ ID NO:7中，第6位氨基酸残基是脯氨酸或色氨酸；第8位氨基酸残基是丙氨酸、亮氨酸或异亮氨酸；第10位和第104位氨基酸各自独立为丝氨酸或苏氨酸；优选地，所述分离的多肽如SEQ ID NO:4所示。
  3. 一种分离的多肽，其特征在于，所述多肽与SEQ ID NO:4所示的氨基酸序列具有至少80％，优选至少85％，更优选至少90％，更优选至少95％，更优选至少97％，更优选至少98％，更优选至少99％的序列同一性，且所述分离多肽在对应于SEQ ID NO:4第6、8、10和104位上的氨基酸残基分别是脯氨酸、缬氨酸、丝氨酸和丝氨酸；优选的，所述多肽获自枯草芽孢杆菌(Bacillus subtilis)。
  4. 一种多核苷酸序列，选自：
    (1)编码权利要求1-3中任一项所述多肽的多核苷酸序列；和
    (2)(1)所述多核苷酸序列的互补序列；和
    (3)(1)或(2)所述序列的长15－30个碱基的片段；
    优选地，所述多核苷酸序列如SEQ ID NO:3所示。
  5. 一种核酸构建体，其特征在于，该核酸构建体包含权利要求4所述的多核苷酸序列和一条或多条与权利要求4所述的多核苷酸序列操作性连接的调控序列；优选地，所述核酸构建体为载体；更优选地，所述载体为表达载体或克隆载体。
  6. 一种遗传工程化的宿主细胞，其特征在于，所述宿主细胞含有权利要求 4所述的多核苷酸序列或权利要求5所述的核酸构建体。
  7. 一种组合物，其特征在于，所述组合物含有权利要求1-3中任一项所述的多肽和任选的辅料，优选地，所述辅料为选自活性炭、氧化铝、硅藻土、多孔陶瓷、多孔玻璃的吸附材料。
  8. 权利要求1-3中任一项所述的多肽、权利要求4所述的多核苷酸序列、权利要求5所述的核酸构建体、权利要求6所述的宿主细胞或权利要求7所述的组合物在油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业中的应用。
  9. 一种酶法脱胶方法，其特征在于，所述方法包括使用权利要求1-3中任一项所述的多肽进行脱胶；优选的，所述方法包括将权利要求1-3中任一项所述的多肽与毛油接触的步骤；优选的，所述步骤包括将所述多肽与50-70℃毛油接触。
  10. 如权利要求9所述的方法，其特征在于，所述方法包括以下特征中的一项或多项：
    (1)以毛油重量计，所述多肽的添加量为10～1000ppm，优选为50～500ppm，更优选为100～300ppm；
    (2)所述脱胶包括：在50～60℃下搅拌1～3小时，然后升温至80～90℃保持1～10分钟；和
    (3)所述毛油选自：大豆油、葵花籽油、花生油、菜籽油、米糠油、玉米油、橄榄油、棕榈油、棕榈仁油、棕榈软脂、卡诺拉油、蓖麻油、椰子油、芫荽油、棉籽油、榛子油、***籽油、亚麻籽油、芒果仁油、白芒花油、牛蹄油、红花油、山茶花油、妥尔油和椿油。
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