WO2020135658A1 - 具有磷脂酶c活性的多肽及其用途 - Google Patents

具有磷脂酶c活性的多肽及其用途 Download PDF

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WO2020135658A1
WO2020135658A1 PCT/CN2019/128973 CN2019128973W WO2020135658A1 WO 2020135658 A1 WO2020135658 A1 WO 2020135658A1 CN 2019128973 W CN2019128973 W CN 2019128973W WO 2020135658 A1 WO2020135658 A1 WO 2020135658A1
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amino acid
seq
polypeptide
acid sequence
phospholipase
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PCT/CN2019/128973
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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 EP19905362.0A priority Critical patent/EP3904510A4/en
Priority to US17/419,082 priority patent/US20220073889A1/en
Publication of WO2020135658A1 publication Critical patent/WO2020135658A1/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)
    • 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
    • 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
    • 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)

Definitions

  • the present application provides polypeptides with phospholipase C activity and uses thereof.
  • Degumming is an important step in oil refining.
  • the traditional hydration degumming method has high economic cost, large energy consumption, and heavy environmental pollution. Therefore, in recent years, a lot of work has been devoted to the use of enzymatic degumming in the degumming process in oil refining.
  • enzymatic degumming can increase economic benefits, achieve energy saving and emission reduction, less pollution to the ecological environment, and has greater advantages in environmental protection, economy, and quality.
  • An enzyme used in the degumming of oils and fats is phospholipase.
  • PLC phospholipase C
  • PLC phospholipase C shows greater advantages, such as increasing the yield of diglyceride (DAG) and reducing the loss of oil.
  • BC-PC-PLC Phosphatidylcholine-specific phospholipase C
  • BC-PC-PLC has a total length of 283 amino acids, including a 24 amino acid signal peptide and a 14 amino acid leader peptide, and a mature peptide of 245 amino acids (see, for example, Johansen, T., Holm, T., Guddal, PH, Sletten, K., Haugli, FB, Little, C. (1988). "Cloning and sequencing" of the gene encoding the phosphoatidylcholine-preferring phospholipase C Bacillus Cereus.” Gene 65(2):293-304).
  • BC-PC-PLC The crystal structure of BC-PC-PLC has been reported. It consists of multiple helical domains, the catalytic site is aspartic acid 55, and contains at least three Zn 2+ binding sites (see, for example, Hough., E., Hansen, LK, Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E., Derewenda, Z. (1989) "High-resolution (1.5 A)crystal structure of phospholipase C from Bacillus cereus.”Nature.338:357-60).
  • BC-PC-PLC Bacillus subtilis and pichia pastoris
  • Durban, MA, Silbersack, J., Schweder, T., Schauer, F., Bornscheuer, UT High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis.
  • the currently reported phospholipase C has low thermal stability and cannot tolerate more than 60°C, so that its optimum degumming temperature needs to be controlled at 50°C, so it has certain limitations in industrial applications. If it can effectively improve the thermal stability of phospholipase C, it is conducive to the industrial application of phospholipase C, including: firstly, the temperature is increased when degumming, which is helpful to reduce the viscosity of oil, improve the separation of oil and phospholipids, and reduce the entrainment of phospholipids Grease, which further improves the yield of grease; secondly, there is currently a combination of PLC and PLA1 for deep degumming.
  • the invention patent CN201480017114.5 mentions a new PLA1 whose degumming temperature is 65°C, so it is more resistant
  • the hot PLC is beneficial for joint degumming with PLA1.
  • the temperature of the crude oil during storage, especially in high temperature weather the crude oil temperature will exceed 50 °C, so if the optimal reaction temperature of the PLC is lower than 50 °C, a lot of Cold water cools the crude oil, which generates a lot of energy. Therefore, the development of new polypeptides with heat-resistant phospholipase C activity has important practical and economic value in this field.
  • the present application provides a polypeptide having phospholipase C activity, the polypeptide having 1) the amino acid sequence shown in SEQ ID No: 2 comprising amino acid substitutions at one or more positions, wherein the one or more Positions selected from the amino acid sequence shown in SEQ ID No: 2 at position 6, position 8, position 10, position 104, position 205, or any combination thereof; or
  • amino acid substitution at position 6 of the amino acid sequence shown in SEQ ID No: 2 is substituted with proline, glycine, hydroxyproline, serine, or threonine for lysine.
  • lysine at position 6 of the amino acid sequence shown in SEQ ID No: 2 is substituted with proline.
  • the amino acid at position 8 of the amino acid sequence shown in SEQ ID No: 2 is substituted with isoleucine, leucine, valine, methionine, alanine, phenylalanine Or norleucine replaces lysine.
  • the lysine at position 8 of the amino acid sequence shown in SEQ ID No: 2 is replaced with isoleucine.
  • the amino acid substitution at position 10 of the amino acid sequence shown in SEQ ID No: 2 is threonine or serine in place of glycine.
  • the glycine at position 10 of the amino acid sequence shown in SEQ ID No: 2 is substituted with threonine.
  • the amino acid substitution at position 104 of the amino acid sequence shown in SEQ ID No: 2 is glycine or proline instead of lysine.
  • the lysine at position 104 of the amino acid sequence shown in SEQ ID No: 2 is substituted with glycine.
  • the amino acid substitution at position 205 of the amino acid sequence shown in SEQ ID No: 2 is tyrosine, tryptophan, phenylalanine, or threonine in place of serine.
  • the serine at position 205 of the amino acid sequence shown in SEQ ID No: 2 is substituted with tyrosine.
  • amino acid sequence of the polypeptide comprises the amino acid sequence shown in SEQ ID No: 4.
  • the amino acid sequence of the polypeptide consists of the amino acid sequence shown in SEQ ID No: 4.
  • the present application provides an isolated polypeptide, wherein the polypeptide has an amino acid sequence shown in SEQ ID NO: 4 of 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 that the isolated polypeptide corresponds to the 6th, 8th, 10th and 10th positions of SEQ ID NO:4
  • the amino acid residues at positions 104 and 205 are proline, isoleucine, threonine, glycine, and tyrosine, respectively.
  • the polypeptide is obtained from Bacillus subtilis.
  • the present application provides a nucleic acid molecule encoding the polypeptide of the first or second aspect.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID No: 3.
  • sequence of the nucleic acid molecule is shown in SEQ ID No: 3.
  • the present application provides a vector comprising the nucleic acid molecule of the third aspect.
  • the vector is an expression vector.
  • the vector is designed for expression in eukaryotic or prokaryotic cells.
  • the vector is designed for expression in bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • the present application provides a cell comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.
  • the cell is a eukaryotic cell or a prokaryotic cell.
  • the cells are bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • the present application provides phospholipase C produced using the cells described in the fifth aspect.
  • the present application provides the polypeptide according to the first aspect or the second aspect, or the polypeptide encoded by the nucleic acid molecule according to the third aspect, or the polypeptide encoded by the vector according to the fourth aspect, or Use of the fermentation broth, concentrated liquid or polypeptide expressed by the cell according to the fifth aspect, or the phospholipase C according to the sixth aspect as the phospholipase C.
  • the use is in a grease degumming process.
  • the present application provides an enzyme composition comprising the polypeptide according to the first aspect or the second aspect, or the polypeptide encoded by the nucleic acid molecule according to the third aspect, or the fourth aspect The polypeptide encoded by the vector, or the polypeptide expressed by the cell according to the fifth aspect, or the phospholipase C according to the sixth aspect, and at least one degumming enzyme.
  • the at least one degumming enzyme is selected from the group consisting of: phospholipase A 1 , phospholipase A 2 , phospholipase B, phospholipase D, pectinase, and mannanase.
  • the present application provides the polypeptide according to the first aspect or the second aspect, or the nucleic acid molecule according to the third aspect, or the vector according to the fourth aspect, or according to the fifth aspect Use of cells or the enzyme composition according to the eighth aspect in the preparation of degumming enzymes.
  • Figure 1 is a standard curve for the determination of phospholipase activity.
  • Figure 2 is a comparison of the thermal stability of PLC-9-49 and PLC-N63DN131SN134D-Y56H, where the circle represents PLC-9-49 and the triangle represents PLC-N63DN131SN134D-Y56H.
  • Figure 3 shows the comparison of the DAG increment of PLC-9-49 and PLC-N63DN131SN134D-Y56H at 55°C and 60°C, respectively, relative to crude oil.
  • SEQ ID No: 1 is the coding nucleic acid sequence of PLC-N63DN131SN134D-Y56H.
  • SEQ ID No: 2 is the amino acid sequence of PLC-N63DN131SN134D-Y56H.
  • SEQ ID No: 3 is the coding nucleic acid sequence of PLC-9-49.
  • SEQ ID No: 4 is the amino acid sequence of PLC-9-49.
  • phosphatidylcholine-specific phospholipase C phosphatidylcholine-phospholipase C
  • phosphatidylcholine-preferring phospholipase C phosphatidylcholine-preferring phospholipase C
  • PC-PLC is used herein to denote phosphatidylcholine-specific phospholipase C or phosphatidylcholine-preferred phospholipase C.
  • BC-PC-PLC phosphatidylcholine-specific phospholipase C of Bacillus cereus
  • BC-PC-PLC may represent wild-type phosphatidylcholine-specific phospholipase C of Bacillus cereus, and may also represent the wild-type phosphatidylcholine-specific phospholipase C in this application. The obtained mutant.
  • amino acid position is represented by a number in this article, the number refers to the amino acid position in SEQ ID No: 2, which is a phosphatidylcholine-specific phospholipase C mutant of Bacillus cereus The amino acid sequence of PLC-N63DN131SN134D-Y56H.
  • polypeptide As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably and refer to a polymer formed by connecting multiple amino acids through peptide bonds. Amino acids can be naturally occurring or synthetic analogs.
  • nucleic acid and “polynucleotide” are used interchangeably, including but not limited to DNA, RNA, and the like. Nucleotides can be naturally occurring or synthetic analogs.
  • the cells herein may be eukaryotic cells or prokaryotic cells, such as, but not limited to, bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • a “conservative substitution” of a specific amino acid sequence refers to the substitution of those amino acids that are not critical to protein activity, or have similar properties (eg, acidic, basic, positively or negatively charged, polar or non-polar, etc.) ) Other amino acids to replace amino acids, so that even the substitution of key amino acids will not significantly change the activity.
  • random saturation mutation refers to designating the site to be mutated in the PCR primer as the NNK merger codon, so as to cover all 20 amino acids to achieve the effect of saturation mutation, and due to the selection of multiple sites Mutation, whose combination is random, is called random saturation mutation.
  • alanine is valine (Val)*, leucine (Leu), and isoleucine (Ile);
  • arginine lysine (Lys)*, glutamine (Gln), and asparagine (Asn);
  • asparagine Asparagine
  • glutamine Gln
  • His histidine
  • Lys lysine
  • Arg arginine
  • Asp aspartic acid
  • aspartic acid Aspartic acid (Asp) are glutamic acid (G1u)* and asparagine (Asn);
  • cysteine is serine (Ser);
  • Gln glutamine
  • G1u glutamic acid
  • Asp aspartic acid
  • Ga ⁇ -hydroxyglutamic acid
  • His histidine
  • isoleucine isoleucine (Leu)*, valine (Val), methionine (Met), alanine (Ala), phenylalanine (Phe), positive Leucine (Nle);
  • leu leucine
  • Nle norleucine
  • Ile isoleucine
  • Val valine
  • Met methionine
  • Met methionine
  • Al alanine
  • Phe benzene Alanine
  • lysine is arginine (Arg)*, glutamine (Gln), asparagine (Asn), ornithine (Orn);
  • the conservative substitutions of methionine (Met) are leucine (Leu)*, isoleucine (Ile), phenylalanine (Phe), and norleucine (Nle);
  • phenylalanine are leucine (Leu)*, valine (Val), isoleucine (Ile), and alanine (Ala);
  • proline glycine (Gly)*, hydroxyproline (Hyp), serine (Ser), threonine (Thr);
  • Thr threonine
  • Ser serine
  • Trp tryptophan
  • Tyr tyrosine
  • Tr tyrosine
  • Phe phenylalanine
  • Thr threonine
  • Ser serine
  • valine Val
  • isoleucine Ile
  • leucine Leu
  • methionine Met
  • phenylalanine Phe
  • alanine Al
  • positive Leucine Nle
  • * represents a preferred conservative substitution.
  • 201480017114.5 relates to a new polypeptide with phospholipase A activity, and its optimal degumming temperature is about 65°C (see paragraph [0173] of patent application CN105073985A), and this PLA1
  • the PLC can also match this degumming temperature.
  • the temperature of the crude oil during storage, especially in high temperature weather will exceed 50°C. Therefore, if the optimal reaction temperature of the PLC is lower than 50°C, cold water is also required to cool the crude oil, which will produce energy Consume.
  • the present application provides a polypeptide having phospholipase C activity, the polypeptide having 1) the amino acid sequence shown in SEQ ID No: 2 comprising amino acid substitutions at one or more positions, wherein the one or more The positions are selected from the amino acid sequence shown in SEQ ID No: 2 at position 6, position 8, position 10, position 104, position 205 or any combination thereof; 2) and 1) 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 6th, 8th, 10th, At least one of the 104th and 205th positions is different from the 6th, 8th, 10th, 104th, and 205th positions in the amino acid sequence shown in SEQ ID No: 2.
  • amino acid at position 6 of the amino acid sequence shown in SEQ ID No: 2 undergoes amino acid substitution.
  • amino acid at position 8 of the amino acid sequence shown in SEQ ID No: 2 undergoes amino acid substitution.
  • amino acid at position 10 of the amino acid sequence shown in SEQ ID No: 2 undergoes amino acid substitution.
  • the amino acid at position 104 of the amino acid sequence shown in SEQ ID No: 2 undergoes an amino acid substitution.
  • the amino acid at position 205 of the amino acid sequence shown in SEQ ID No: 2 undergoes an amino acid substitution.
  • amino acids at positions 6 and 8 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6 and 10 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acid substitutions occur at amino acids 6 and 104 of the amino acid sequence shown in SEQ ID No: 2.
  • amino acids at positions 6 and 205 of the amino acid sequence shown in SEQ ID No: 2 undergo amino acid substitutions.
  • amino acid substitutions occur at the 8th and 10th amino acids of the amino acid sequence shown in SEQ ID No: 2.
  • amino acid substitutions occur at the 8th and 104th amino acids of the amino acid sequence shown in SEQ ID No: 2.
  • amino acid substitutions occur at the 8th and 205th amino acids of the amino acid sequence shown in SEQ ID No: 2.
  • amino acid substitutions occur at amino acids 10 and 104 of the amino acid sequence shown in SEQ ID No: 2.
  • amino acid substitutions occur at amino acids 10 and 205 of the amino acid sequence shown in SEQ ID No: 2.
  • amino acids at positions 104 and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6, 8 and 10 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6, 8 and 104 of the amino acid sequence shown in SEQ ID No: 2 are subjected to amino acid substitutions.
  • amino acids at positions 6, 8, and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6, 10, and 104 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6, 10, and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6, 104, and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 8, 10, and 104 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 8, 10, and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 8, 104, and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acid substitutions occur at amino acids 10, 104, and 205 of the amino acid sequence shown in SEQ ID No: 2.
  • amino acids at positions 6, 8, 10, and 104 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6, 8, 10, and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acids at positions 6, 8, 104, and 205 of the amino acid sequence shown in SEQ ID No: 2 have amino acid substitutions.
  • amino acid substitutions occur at the amino acids at positions 6, 10, 104, and 205 of the amino acid sequence shown in SEQ ID No: 2.
  • amino acid substitutions are made at the 8th, 10th, 104th, and 205th amino acids of the amino acid sequence shown in SEQ ID No: 2.
  • amino acid substitution at position 6 of the amino acid sequence shown in SEQ ID No: 2 is substituted with proline, glycine, hydroxyproline, serine, or threonine for lysine.
  • lysine at position 6 of the amino acid sequence shown in SEQ ID No: 2 is substituted with proline.
  • the amino acid at position 8 of the amino acid sequence shown in SEQ ID No: 2 is substituted with isoleucine, leucine, valine, methionine, alanine, phenylalanine Or norleucine replaces lysine.
  • the lysine at position 8 of the amino acid sequence shown in SEQ ID No: 2 is replaced with isoleucine.
  • the amino acid substitution at position 10 of the amino acid sequence shown in SEQ ID No: 2 is threonine or serine in place of glycine.
  • the glycine at position 10 of the amino acid sequence shown in SEQ ID No: 2 is substituted with threonine.
  • the amino acid substitution at position 104 of the amino acid sequence shown in SEQ ID No: 2 is glycine or proline instead of lysine.
  • the lysine at position 104 of the amino acid sequence shown in SEQ ID No: 2 is substituted with glycine.
  • the amino acid substitution at position 205 of the amino acid sequence shown in SEQ ID No: 2 is tyrosine, tryptophan, phenylalanine, or threonine in place of serine.
  • the serine at position 205 of the amino acid sequence shown in SEQ ID No: 2 is substituted with tyrosine.
  • amino acid sequence of the polypeptide comprises the amino acid sequence shown in SEQ ID No: 4.
  • the amino acid sequence of the polypeptide consists of the amino acid sequence shown in SEQ ID No: 4.
  • the present application also considers functional variants of the polypeptides described in the first aspect.
  • the functional variant is a conservative substitution variant.
  • the present application provides an isolated polypeptide, wherein the polypeptide has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acid sequence shown in SEQ ID NO:4 , More preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity, and the isolated polypeptide corresponds to the 6th, 8th, and 10th positions of SEQ ID NO:4,
  • the amino acid residues at positions 104 and 205 are proline, isoleucine, threonine, glycine, and tyrosine, respectively.
  • the polypeptide is obtained from Bacillus subtilis.
  • the polypeptide has at least 97%, at least 98%, at least 99%, at least 99.5%, or higher sequence identity with the amino acid sequence shown in SEQ ID NO:4.
  • the polypeptide has at least 97.2%, at least 97.6%, at least 98%, at least 98.4%, at least 98.8%, at least 99.2%, at least 99.6%, or more than the amino acid sequence shown in SEQ ID NO:4 High sequence identity.
  • the amino acid residue at position 6 corresponding to SEQ ID NO: 4 of the isolated polypeptide is proline.
  • the amino acid residue at position 8 corresponding to SEQ ID NO: 4 of the isolated polypeptide is isoleucine.
  • the amino acid residue at position 10 corresponding to SEQ ID NO: 4 of the isolated polypeptide is threonine.
  • the amino acid residue at position 104 corresponding to SEQ ID NO: 4 of the isolated polypeptide is glycine.
  • the amino acid residue at position 205 corresponding to SEQ ID NO: 4 of the isolated polypeptide is tyrosine.
  • the amino acid residue at position 6 of the isolated polypeptide corresponding to SEQ ID NO: 4 is proline, and/or the isolated polypeptide corresponds to the amino acid residue of SEQ ID NO: 4
  • the amino acid residue at position 8 is isoleucine, and/or the amino acid residue at position 10 corresponding to SEQ ID NO: 4 of the isolated polypeptide is threonine, and/or the separation
  • the amino acid residue of the polypeptide corresponding to position 104 of SEQ ID NO: 4 is glycine, and/or the amino acid residue of the isolated polypeptide corresponding to position 205 of SEQ ID NO: 4 is tyrosine acid.
  • the present application provides a nucleic acid molecule encoding the polypeptide of the first or second aspect.
  • the present application considers different nucleic acid molecules that can be obtained due to the degeneracy of the genetic code or the preference of different species for the code.
  • the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID No: 3.
  • sequence of the nucleic acid molecule is shown in SEQ ID No: 3.
  • the present application provides a vector comprising the nucleic acid molecule of the third aspect.
  • the vector is an expression vector.
  • the vector is designed for expression in eukaryotic or prokaryotic cells.
  • the vector is designed for expression in bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • the vector is a plasmid.
  • Suitable eukaryotic cell or prokaryotic cell vectors are well known to those skilled in the art, and various maternal vectors are commercially available.
  • the carrier include, but are not limited to, various carriers used in the embodiments of the present application.
  • the present application provides a cell comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.
  • the cell is a eukaryotic cell or a prokaryotic cell.
  • the cells are bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • the cells are pichia pastoris cells.
  • the cell is a Bacillus subtilis cell.
  • the cell is an E. coli cell.
  • the nucleic acid molecule may be located extrachromosomally (for example, in a vector) or may be integrated into the chromosome of the host cell.
  • Techniques for integrating nucleic acid molecules into the chromosome of the host cell and introducing the vector into the host cell by transformation or transfection are well known to those skilled in the art.
  • the present application provides phospholipase C produced using the cells described in the fifth aspect.
  • Techniques for producing target polypeptides or proteins using genetically engineered host cells are well known to those skilled in the art.
  • the present application provides the polypeptide according to the first aspect or the second aspect, or the polypeptide encoded by the nucleic acid molecule according to the third aspect, or the polypeptide encoded by the vector according to the fourth aspect, or Use of the fermentation broth, concentrated liquid or polypeptide expressed by the cell according to the fifth aspect, or the phospholipase C according to the sixth aspect as phospholipase C.
  • the use is in a grease degumming process.
  • phosphatidylcholine-specific phospholipase C in the process of degumming oils and fats is known in the art.
  • Phospholipase C can hydrolyze the phospholipid of the colloidal component in the oil to generate a hydrophilic phosphoric acid part and a lipophilic DAG.
  • the hydrophilic part is taken away by water to remove the colloidal part.
  • DAG increases the oil yield.
  • the enzymatic degumming process involves heating the raw oil to 60°C, adding the phospholipase C solution, mixing at high speed, stirring and reacting in the reactor for 2 hours, and then centrifuging to separate the water phase and the oil phase.
  • the present application provides an enzyme composition comprising the polypeptide according to the first aspect or the second aspect, or the polypeptide encoded by the nucleic acid molecule according to the third aspect, or the fourth aspect The polypeptide encoded by the vector, or the polypeptide expressed by the cell according to the fifth aspect, or the phospholipase C according to the sixth aspect, and at least one degumming enzyme.
  • the at least one degumming enzyme is selected from the group consisting of: phospholipase A 1 , phospholipase A 2 , phospholipase B, phospholipase D, pectinase, and mannanase.
  • the enzyme composition includes the polypeptide of the first or second aspect and at least one degumming enzyme.
  • the enzyme composition includes the polypeptide encoded by the nucleic acid molecule of the third aspect and at least one degumming enzyme.
  • the enzyme composition includes the polypeptide encoded by the vector of the fourth aspect and at least one degumming enzyme.
  • the enzyme composition includes the cell-expressed polypeptide as described in the fifth aspect and at least one degumming enzyme.
  • the enzyme composition includes phospholipase C as described in the sixth aspect and at least one degumming enzyme.
  • the enzyme composition includes the polypeptide according to the first aspect or the second aspect, and/or the polypeptide encoded by the nucleic acid molecule according to the third aspect, and/or the vector according to the fourth aspect The encoded polypeptide, and/or the cell-expressed polypeptide of the fifth aspect, and/or the phospholipase C of the sixth aspect, and at least one degumming enzyme.
  • the present application provides the polypeptide according to the first aspect or the second aspect, or the nucleic acid molecule according to the third aspect, or the vector according to the fourth aspect, or according to the fifth aspect Use of cells or the enzyme composition according to the eighth aspect in the preparation of degumming enzymes.
  • Pichia pastoris SMD1168 Invitrogen, Catalog No. C17500
  • E. coli DH5 ⁇ TAKARA, Catalog No. 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 and no amino acids), 1% glycerin, 1M sorbitol, 4 ⁇ 10-5% D-biotin, 2% agar.
  • yeast nitrogen source base YNB
  • yeast nitrogen source base YNB
  • yeast nitrogen source base YNB
  • yeast nitrogen source base YNB
  • Table 1 Amount of reagent added when drawing a standard curve for determination of phospholipase activity.
  • the solution was treated at 37°C for 15 minutes, and then 500 ⁇ l of 0.5 N NaOH was added, and the absorbance was measured at 410 nm.
  • Sample enzyme activity (U/ml) A (410nm absorbance) ⁇ 0.1935 ⁇ dilution factor ⁇ 10/15
  • DNA polymerase DNA polymerase (purchased from Bao Bioengineering (Dalian) Co., Ltd.);
  • T4 DNA ligase (purchased from Fuzymes Co., Ltd.).
  • Example 1 Construction and screening of phospholipase C mutant library
  • the pmAO-PLC-N63DN131SN134D-Y56H carrier was prepared according to the method described in the Chinese invention application CN201510946696.1.
  • pmAO-PLC-N63DN131SN134D-Y56H vector as a template, construct a library of random saturation mutations at the 6th, 8th, 10th, 104th, and 205th amino acids (commissioned by Suzhou Hongxun Biotechnology Co., Ltd.) Library construction).
  • the plasmid library was transformed into E. coli DH5 ⁇ strain, and all the obtained E. coli clones were washed into LB liquid medium (containing 100 ⁇ g/ml ampicillin), and cultured at 37°C for 4 hours.
  • the plasmid was extracted, linearized with SalI, and a fragment of about 8.5 kb was recovered. Take 500 ng of vector and transform the vector into competent cells of Pichia pastoris M314 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 inoculate on the BMM-soybean phospholipid screening plate. Select clones with large white precipitation circles. The mutant strain was obtained and designated as PLC-9-49.
  • the PLC-9-49 strain was inoculated in 3ml YPD liquid medium and cultured at 30°C overnight to extract genomic DNA. Using the genomic DNA of PLC-9-49 strain as a template, use DNA polymerase and primers were used to PCR amplify AOX1-5/AOX1-3 to obtain the PLC DNA sequence of PLC-9-49 strain. among them,
  • primer AOX1-5 The sequence of primer AOX1-5 is: 5'-GACTGGTTCCAATTGACAACG-3';
  • primer AOX1-3 The sequence of primer AOX1-3 is: 5'-GGCAAATGGCATTCTGACATCCTC-3'.
  • the obtained sequence was sent to Shanghai Biotech Engineering Co., Ltd., and the primers were used to sequence AOX1-5/AOX1-3.
  • the DNA sequencing result of PLC-9-49 is shown in SEQ ID No: 3.
  • SEQ ID No: 3 After comparison, it was found that compared with SEQ ID No: 1, there are several base mutations in SEQ ID No: 3, which makes the lysine at position 6 in SEQ ID No: 2 mutated to proline, Lysine at position 8 is mutated to isoleucine, glycine at position 10 to threonine, lysine at position 104 to glycine, and serine at position 205 to tyrosine.
  • the amino acid sequence of PLC-9-49 is shown in SEQ ID No: 4.
  • 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 ultrafiltration concentrated fermentation broth was incubated at 60°C, 65°C, 70°C and 75°C for 2 hours, 0.5 ⁇ l of the fermentation broth concentrate was added to 600 ⁇ l of pNPPC reaction buffer, reacted at 37°C for 15min, and then 500 ⁇ l was added The reaction was stopped with 0.5N NaOH, and the absorbance was measured at 410nm. According to the standard curve, the phospholipase C activity of each fermentation broth sample was calculated.
  • the degumming temperature of PLC-9-49 is about 5°C higher than that of PLC-N63DN131SN134D-Y56H, so the industrial applicability is better.

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Abstract

提供具有磷脂酶C活性的多肽，该多肽具有1)包含在一个或多个位置发生氨基酸取代的SEQ ID No:2所示的氨基酸序列，其中所述一个或多个位置选自SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位或其任意组合；或2)与1)至少80％序列同一性，且第6位、第8位、第10位、第104位、第205位的至少1个位置与SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位不同。提供编码该多肽的核酸分子、包含核酸分子的载体以及包含核酸分子或载体的细胞。提供多肽、核酸分子载体以及细胞的用途。

Description

具有磷脂酶C活性的多肽及其用途 技术领域
本申请提供了具有磷脂酶C活性的多肽及其用途。
背景技术
脱胶是油脂精炼的重要步骤，传统的水化脱胶法经济成本高，物料能耗大，环境污染重，所以近些年来，很多工作致力于将酶法脱胶用于油脂精炼中的脱胶环节。同传统方法相比，酶法脱胶能够提高经济效益，实现节能减排，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。油脂脱胶中所用的一种酶为磷脂酶。同其他脱胶酶相比，磷脂酶C(PLC)表现出更大的优势，例如，增加甘二酯(DAG)的得率，以及减少得油量的损失。
蜡样芽胞杆菌(Bacillus cereus)的磷脂酰胆碱特异性磷脂酶C(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(参见，例如Johansen,T.,Holm,T.,Guddal,P.H.,Sletten,K.,Haugli,F.B.,Little,C.(1988)."Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus."Gene 65(2):293-304)。
先前有文献报道，野生型BC-PC-PLC的活性位点为Glu4、Asp55、Tyr56、Glu146、Ser64、Thr65、Phe66、Phe70、Ile80、Thr133、Asn134、Leu135、Ser143(参见，例如Hough.,E.,Hansen,L.K.,Birknes,B.,Jynge,K.,Hansen,S.,Hordvik,A.,Little,C.,Dodson,E.,Derewenda,Z.(1989)"High-resolution(1.5A)crystal structure of phospholipase C from Bacillus cereus."Nature.338:357-60)。
BC-PC-PLC的晶体结构已有报道，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Zn 2+结合位点(参见，例如 Hough.,E.,Hansen,L.K.,Birknes,B.,Jynge,K.,Hansen,S.,Hordvik,A.,Little,C.,Dodson,E.,Derewenda,Z.(1989)"High-resolution(1.5A)crystal structure of phospholipase C from Bacillus cereus."Nature.338:357-60)。BC-PC-PLC的异源表达研究较少，已有报道涉及在枯草芽孢杆菌(Bacillus subtilis)和毕赤酵母(pichia pastoris)中表达BC-PC-PLC(参见，例如Durban,M.A.,Silbersack,J.,Schweder,T.,Schauer,F.,Bornscheuer,U.T.(2007)High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillussubtilis.Appl Microbiol Biotechnol 74(3):634-639；以及Seo,K.H,Rhee J.I.(2004)High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization.Biotechnol Lett 26(19):1475-1479)。
但现有报道的磷脂酶C的热稳定性不高，不能耐受超过60℃，使其最适的脱胶温度需要控制在50℃，因而使其在工业应用中具有一定的局限性。如能有效提高磷脂酶C的热稳定性，有利于磷脂酶C的工业应用，具体包括：首先脱胶时将温度提高，有利于降低油的粘度，提高油脂和磷脂的分离程度，减少磷脂中夹带的油脂，进一步提高油脂的得率；其次目前有采用PLC与PLA1联合使用进行深度脱胶，发明专利CN201480017114.5中就提到了一种新的PLA1其最适的脱胶温度在65℃，因此更耐热的PLC有利于与PLA1进行联合脱胶，另外，有时毛油在储藏过程中特别是在高温天气，毛油的温度会超过50℃，因此如果PLC的最适反应温度低于50℃还需要大量冷水进行对毛油降温，产生大量能耗。因此，开发新的具有耐热性能的磷脂酶C活性的多肽对于本领域具有重要实用和经济价值。
发明概述
第一方面，本申请提供了具有磷脂酶C活性的多肽，所述多肽具有1)包含在一个或多个位置发生氨基酸取代的SEQ ID No:2所示的氨基酸序列，其中所述一个或多个位置选自SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位或其任意组合；或
2)与1)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，且第6位、第8位、第10位、第104位、第205位的至少1个位置与SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位不同。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位和第205位全部发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位的氨基酸取代为脯氨酸、甘氨酸、羟脯氨酸、丝氨酸或苏氨酸取代赖氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位的赖氨酸被脯氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位的氨基酸取代为异亮氨酸、亮氨酸、缬氨酸、甲硫氨酸、丙氨酸、苯丙氨酸或正亮氨酸取代赖氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位的赖氨酸被异亮氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位的氨基酸取代为苏氨酸或丝氨酸取代甘氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位的甘氨酸被苏氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第104位的氨基酸取代为甘氨酸或脯氨酸取代赖氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第104位的赖氨酸被甘氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第205位的氨基酸取代为酪氨酸、色氨酸、苯丙氨酸、或苏氨酸取代丝氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第205位的丝氨酸被酪氨酸取代。
在一些实施方案中，所述多肽的氨基酸序列包含如SEQ ID No:4 所示的氨基酸序列。
在一些实施方案中，所述多肽的氨基酸序列由如SEQ ID No:4所示的氨基酸序列组成。
第二方面，本申请提供了一种分离的多肽，其中所述多肽与SEQ ID NO:4所示的氨基酸序列具有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，并且所述分离的多肽在对应于SEQ ID NO:4的第6位、第8位、第10位、第104位和第205位上的氨基酸残基分别是脯氨酸、异亮氨酸、苏氨酸、甘氨酸和酪氨酸。优选的，所述多肽获得自枯草芽孢杆菌(Bacillus subtilis)。
第三方面，本申请提供了编码第一方面或第二方面所述的多肽的核酸分子。
在一些实施方案中，所述核酸分子包含如SEQ ID No:3所示的核酸序列。
在一些实施方案中，所述核酸分子的序列如SEQ ID No:3所示。
第四方面，本申请提供了包含第三方面所述的核酸分子的载体。
在一些实施方案中，所述载体为表达载体。
在一些实施方案中，所述载体被设计用于真核细胞或原核细胞中表达。
在一些实施方案中，所述载体被设计用于细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞中表达。
第五方面，本申请提供了包含第三方面所述的核酸分子或第四方面所述的载体的细胞。
在一些实施方案中，所述细胞为真核细胞或原核细胞。
在一些实施方案中，所述细胞为细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞。
第六方面，本申请提供了使用第五方面所述的细胞产生的磷脂酶C。
第七方面，本申请提供了如第一方面或第二方面所述的多肽、或如第三方面所述的核酸分子编码的多肽、或如第四方面所述的载体编 码的多肽、或如第五方面所述的细胞表达的发酵液、浓缩液或多肽、或如第六方面所述的磷脂酶C作为磷脂酶C的用途。
在一些实施方案中，所述用途为在油脂脱胶工艺中的用途。
第八方面，本申请提供了一种酶组合物，其包括如第一方面或第二方面所述的多肽、或如第三方面所述的核酸分子编码的多肽、或如第四方面所述的载体编码的多肽、或如第五方面所述的细胞表达的多肽、或如第六方面所述的磷脂酶C，以及至少一种脱胶酶。
在一些实施方案中，至少一种脱胶酶选自：磷脂酶A 1、磷脂酶A 2、磷脂酶B、磷脂酶D、果胶酶和甘露聚糖酶。
第九方面，本申请提供了如第一方面或第二方面所述的多肽、或如第三方面所述的核酸分子、或如第四方面所述的载体、或如第五方面所述的细胞、或如第八方面所述的酶组合物在制备脱胶酶中的用途。
附图说明
图1为磷脂酶活力测定标准曲线。
图2为PLC-9-49和PLC-N63DN131SN134D-Y56H的热稳定性比较，其中圆圈表示PLC-9-49，三角表示PLC-N63DN131SN134D-Y56H。
图3为PLC-9-49和PLC-N63DN131SN134D-Y56H分别在55℃和60℃的条件下进行脱胶，相对于毛油的DAG增量的比较。
序列说明
SEQ ID No:1为PLC-N63DN131SN134D-Y56H的编码核酸序列。
SEQ ID No:2为PLC-N63DN131SN134D-Y56H的氨基酸序列。
SEQ ID No:3为PLC-9-49的编码核酸序列。
SEQ ID No:4为PLC-9-49的氨基酸序列。
发明详细描述
定义
本文中所述的磷脂酰胆碱特异性磷脂酶C(specific phosphatidylcholine phospholipase C)与磷脂酰胆碱偏好型磷脂酶C (phosphatidylcholine-preferring phospholipase C)为同义术语，并且也是本领域技术人员能够容易理解的。本文中使用简写PC-PLC来表示磷脂酰胆碱特异性磷脂酶C或磷脂酰胆碱偏好型磷脂酶C。
本文中使用的磷脂酰胆碱特异性磷脂酶C的一个实例为蜡样芽胞杆菌(Bacillus cereus)的磷脂酰胆碱特异性磷脂酶C，在本文中以简写BC-PC-PLC表示。应当理解，在本文中，BC-PC-PLC可表示蜡样芽胞杆菌的野生型磷脂酰胆碱特异性磷脂酶C，可也表示本申请中基于该野生型磷脂酰胆碱特异性磷脂酶C获得的突变体。
在本文中涉及用数字表示氨基酸位置的情况中，所述数字是参照SEQ ID No:2中的氨基酸位置，SEQ ID No:2为蜡样芽胞杆菌的磷脂酰胆碱特异性磷脂酶C突变体PLC-N63DN131SN134D-Y56H的氨基酸序列。
本文中使用了国际通用的氨基酸的单字母或三字母缩写。
本文所用的术语“多肽”、“肽”和“蛋白”可互换使用，表示多个氨基酸通过肽键连接形成的聚合物。氨基酸可以是天然存在的或人工合成的类似物。
本文所用的术语“核酸”和“多核苷酸”可互换使用，包括但不限于DNA、RNA等。核苷酸可以是天然存在的或人工合成的类似物。
本文中的细胞可以是真核细胞或原核细胞，例如，但不限于细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞。
本文所用的术语“保守型取代”指蛋白的氨基酸组成的变化，所述变化不会显著改变蛋白的活性。因此，具体氨基酸序列的“保守型取代”指对蛋白活性并非关键的那些氨基酸的取代，或用具有相似性质(例如酸性、碱性、带正电荷或带负电荷、极性或非极性等)的其它氨基酸取代氨基酸，使得即便是关键氨基酸的取代也不会显著改变活性。
本文所用的术语“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，因此称之为随机饱和突变。
提供功能上相似的氨基酸的保守型取代表在本领域中是熟知的。例如，以下列出了各个氨基酸的可选的保守氨基酸取代：
丙氨酸(Ala)的保守取代为缬氨酸(Val)*、亮氨酸(Leu)、异亮氨酸(Ile)；
精氨酸(Arg)的保守取代为赖氨酸(Lys)*、谷氨酰胺(Gln)、天冬酰胺(Asn)；
天冬酰胺(Asn)的保守取代为谷氨酰胺(Gln)*、组氨酸(His)、赖氨酸(Lys)、精氨酸(Arg)、天冬氨酸(Asp)；
天冬氨酸(Asp)的保守取代为谷氨酸(G1u)*、天冬酰胺(Asn)；
半胱氨酸(Cys)的保守取代为丝氨酸(Ser)；
谷氨酰胺(Gln)的保守取代为天冬酰胺(Asn)*、组氨酸(His)、赖氨酸(Lys)；
谷氨酸(G1u)的保守取代为天冬氨酸(Asp)*、γ-羟基谷氨酸(Gla)；
甘氨酸(Gly)的保守取代为脯氨酸(Pro)；
组氨酸(His)的保守取代为天冬酰胺(Asn)、谷氨酰胺(Gln)、赖氨酸(Lys)、精氨酸(Arg)*；
异亮氨酸(Ile)的保守取代为亮氨酸(Leu)*、缬氨酸(Val)、甲硫氨酸(Met)、丙氨酸(Ala)、苯丙氨酸(Phe)、正亮氨酸(Nle)；
亮氨酸(Leu)的保守取代为正亮氨酸(Nle)、异亮氨酸(Ile)*、缬氨酸(Val)、甲硫氨酸(Met)、丙氨酸(Ala)、苯丙氨酸(Phe)；
赖氨酸(Lys)的保守取代为精氨酸(Arg)*、谷氨酰胺(Gln)、天冬酰胺(Asn)、鸟氨酸(Orn)；
甲硫氨酸(Met)的保守取代为亮氨酸(Leu)*、异亮氨酸(Ile)、苯丙氨酸(Phe)、正亮氨酸(Nle)；
苯丙氨酸(Phe)的保守取代为亮氨酸(Leu)*、缬氨酸(Val)、异亮氨酸(Ile)、丙氨酸(Ala)；
脯氨酸(Pro)的保守取代为甘氨酸(Gly)*、羟脯氨酸(Hyp)、丝氨酸(Ser)、苏氨酸(Thr)；
丝氨酸(Ser)的保守取代为苏氨酸(Thr)；
苏氨酸(Thr)的保守取代为丝氨酸(Ser)；
色氨酸(Trp)的保守取代为酪氨酸(Tyr)；
酪氨酸(Tyr)的保守取代为色氨酸(Trp)、苯丙氨酸(Phe)*、苏氨酸(Thr)、丝氨酸(Ser)；
缬氨酸(Val)的保守取代为异亮氨酸(Ile)、亮氨酸(Leu)*、甲硫氨酸(Met)、苯丙氨酸(Phe)、丙氨酸(Ala)、正亮氨酸(Nle)。
其中，*表示优选的保守取代。
此外，还可参阅Creighton,Proteins:Structures and Molecular Properties(蛋白：结构和分子特性),W.H.Freeman and Company,New York(2nd Ed.,1992)。
技术方案详细描述
在本申请发明人先前的专利申请CN 201510946696.1中，将BC-PC-PLC的四个氨基酸(即第56位、第63位、第131位和第134位的氨基酸)分别突变为组氨酸(Y56H)、天冬氨酸(N63D)、丝氨酸(N131S)和天冬氨酸(N134D)，突变后的氨基酸序列如SEQ ID No：2所示，编码序列如SEQ ID No：1所示，本文也称为“PLC-N63DN131SN134D-Y56H”。PLC-N63DN131SN134D-Y56H的比酶活相比于野生型BC-PC-PLC有提高，但此突变体的稳定性不高，不能耐受超过60℃的温度，最合适的脱胶温度为50℃，这使得此突变体在工业应用中具有一定的局限性。首先，在脱胶时，一般会提高温度，这样有利于降低油的粘度，提高油脂和磷脂的分离程度，减少磷脂中夹带的油脂，而进一步提高油脂的得率。其次，目前的技术中可能联合使用PLC与PLA1以进行深度脱胶。例如，在中国专利申请第201480017114.5号中涉及一种新的具有磷脂酶A活性的多肽，其最适的脱胶温度约65℃(参见专利申请CN105073985A的第[0173]段)，而与这样的PLA1进行联合脱胶最好希望PLC也能配合这样的脱胶温度。此外，有时毛油在储藏过程中尤其是在高温天气，其温度会超过50℃，因此，如果PLC的最适反应温度低于50℃，则还需要冷水对毛油进行降温，这将产生能耗。
因此，为了提高磷脂酶C的热稳定性和/或提高磷脂酶C的适用性，本申请的发明人对PLC-N63DN131SN134D-Y56H进行了改进，并建立了本申请的各项发明。
第一方面，本申请提供了具有磷脂酶C活性的多肽，所述多肽具有1)包含在一个或多个位置发生氨基酸取代的SEQ ID No:2所示的 氨基酸序列，其中所述一个或多个位置选自SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位或其任意组合；2)与1)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，且第6位、第8位、第10位、第104位、第205位的至少1个位置与SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位不同。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位和第205位全部发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位和第8位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位和第10位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位和第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位和第10位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位和 第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位和第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第104位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第8位和第10位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第8位和第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第8位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第10位和第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第10位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第104位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位、第10位和第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位、第10位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位、第104位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位、第104位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、 第8位、第10位和第104位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第8位、第10位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第8位、第104位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位、第10位、第104位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位、第10位、第104位和第205位的氨基酸发生氨基酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位的氨基酸取代为脯氨酸、甘氨酸、羟脯氨酸、丝氨酸或苏氨酸取代赖氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第6位的赖氨酸被脯氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位的氨基酸取代为异亮氨酸、亮氨酸、缬氨酸、甲硫氨酸、丙氨酸、苯丙氨酸或正亮氨酸取代赖氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第8位的赖氨酸被异亮氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位的氨基酸取代为苏氨酸或丝氨酸取代甘氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第10位的甘氨酸被苏氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第104位的氨基酸取代为甘氨酸或脯氨酸取代赖氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第104位的赖氨酸被甘氨酸取代。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第205位的氨基酸取代为酪氨酸、色氨酸、苯丙氨酸、或苏氨酸取代丝氨酸。
在一些实施方案中，SEQ ID No:2所示的氨基酸序列的第205位 的丝氨酸被酪氨酸取代。
在一些实施方案中，所述多肽的氨基酸序列包含如SEQ ID No:4所示的氨基酸序列。
在一些实施方案中，所述多肽的氨基酸序列由如SEQ ID No:4所示的氨基酸序列组成。
本申请还考虑了第一方面所述的多肽的功能性变体。在一些实施方案中，所述功能性变体为保守型取代变体。
第二方面，本申请提供了一种分离的多肽，其中所述多肽与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，并且所述分离的多肽在对应于SEQ ID NO:4的第6位、第8位、第10位、第104位和第205位上的氨基酸残基分别是脯氨酸、异亮氨酸、苏氨酸、甘氨酸和酪氨酸。
在一些实施方案中，所述多肽获得自枯草芽孢杆菌(Bacillus subtilis)。
在一些实施方案中，所述多肽与SEQ ID NO:4所示的氨基酸序列具有至少97％、至少98％、至少99％、至少99.5％或更高的序列同一性。
在一些实施方案中，所述多肽与SEQ ID NO:4所示的氨基酸序列具有至少97.2％、至少97.6％、至少98％、至少98.4％、至少98.8％、至少99.2％、至少99.6％或更高的序列同一性。
在一些实施方案中，所述分离的多肽在对应于SEQ ID NO:4的第6位上的氨基酸残基是脯氨酸。
在一些实施方案中，所述分离的多肽在对应于SEQ ID NO:4的第8位上的氨基酸残基是异亮氨酸。
在一些实施方案中，所述分离的多肽在对应于SEQ ID NO:4的第10位上的氨基酸残基是苏氨酸。
在一些实施方案中，所述分离的多肽在对应于SEQ ID NO:4的第104位上的氨基酸残基是甘氨酸。
在一些实施方案中，所述分离的多肽在对应于SEQ ID NO:4的第 205位上的氨基酸残基是酪氨酸。
在一些实施方案中，所述分离的多肽在对应于SEQ ID NO:4的第6位上的氨基酸残基是脯氨酸，和/或所述分离的多肽在对应于SEQ ID NO:4的第8位上的氨基酸残基是异亮氨酸，和/或所述分离的多肽在对应于SEQ ID NO:4的第10位上的氨基酸残基是苏氨酸，和/或所述分离的多肽在对应于SEQ ID NO:4的第104位上的氨基酸残基是甘氨酸，和/或所述分离的多肽在对应于SEQ ID NO:4的第205位上的氨基酸残基是酪氨酸。
第三方面，本申请提供了编码第一方面或第二方面所述的多肽的核酸分子。本申请考虑了由于遗传密码子的简并性或不同物种对于密码子的偏好性所能获得的不同核酸分子。
在一些实施方案中，所述核酸分子包含如SEQ ID No:3所示的核酸序列。
在一些实施方案中，所述核酸分子的序列如SEQ ID No:3所示。
第四方面，本申请提供了包含第三方面所述的核酸分子的载体。
在一些实施方案中，所述载体为表达载体。
在一些实施方案中，所述载体被设计用于真核细胞或原核细胞中表达。
在一些实施方案中，所述载体被设计用于细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞中表达。
在一些实施方案中，所述载体为质粒。
合适的真核细胞或原核细胞载体是本领域技术人员公知的，并且多种母体载体都是可以商业购买的。载体的实例包括但不限于本申请的实施例中使用的多种载体。
第五方面，本申请提供了包含第三方面所述的核酸分子或第四方面所述的载体的细胞。
在一些实施方案中，所述细胞为真核细胞或原核细胞。
在一些实施方案中，所述细胞为细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞。
在一些实施方案中，所述细胞为毕赤酵母(pichia pastoris)细胞。
在一些实施方案中，所述细胞为枯草芽孢杆菌(Bacillus subtilis)细胞。
在一些实施方案中，所述细胞为大肠杆菌细胞。
关于包含本申请的核酸分子的细胞，所述核酸分子可位于染色体外(例如位于载体中)，也可以被整合到宿主细胞的染色体中。将核酸分子整合到宿主细胞的染色体中以及将载体通过转化或转染引入宿主细胞中的技术均是本领域技术人员所公知的。
第六方面，本申请提供了使用第五方面所述的细胞产生的磷脂酶C。利用基因工程化宿主细胞产生目标多肽或蛋白的技术是本领域技术人员所公知的。
第七方面，本申请提供了如第一方面或第二方面所述的多肽、或如第三方面所述的核酸分子编码的多肽、或如第四方面所述的载体编码的多肽、或如第五方面所述的细胞表达的发酵液、浓缩液或多肽、或如第六方面所述的磷脂酶C，其作为磷脂酶C的用途。
在一些实施方案中，所述用途为在油脂脱胶工艺中的用途。
磷脂酰胆碱特异性磷脂酶C在油脂脱胶工艺中的应用是本领已知的。磷脂酶C能够水解油中的胶质成分磷脂，生成亲水的磷酸部分和亲油的DAG，亲水部分被水带走从而去除胶质部分，DAG则增加了油的得率。例如，酶法脱胶过程为将原料油加热至60℃，加入磷脂酶C溶液，高速剪切混合后，在反应器中搅拌反应2h，随后离心分离水相及油相。
第八方面，本申请提供了一种酶组合物，其包括如第一方面或第二方面所述的多肽、或如第三方面所述的核酸分子编码的多肽、或如第四方面所述的载体编码的多肽、或如第五方面所述的细胞表达的多肽、或如第六方面所述的磷脂酶C，以及至少一种脱胶酶。
在一些实施方案中，所述至少一种脱胶酶选自：磷脂酶A 1、磷脂酶A 2、磷脂酶B、磷脂酶D、果胶酶和甘露聚糖酶。
在一些实施方案中，酶组合物包括如第一方面或第二方面所述的多肽以及至少一种脱胶酶。
在一些实施方案中，酶组合物包括如第三方面所述的核酸分子编 码的多肽以及至少一种脱胶酶。
在一些实施方案中，酶组合物包括如第四方面所述的载体编码的多肽以及至少一种脱胶酶。
在一些实施方案中，酶组合物包括如第五方面所述的细胞表达的多肽以及至少一种脱胶酶。
在一些实施方案中，酶组合物包括如第六方面所述的磷脂酶C以及至少一种脱胶酶。
在一些实施方案中，酶组合物包括如第一方面或第二方面所述的多肽，和/或如第三方面所述的核酸分子编码的多肽，和/或如第四方面所述的载体编码的多肽，和/或如第五方面所述的细胞表达的多肽，和/或如第六方面所述的磷脂酶C，以及至少一种脱胶酶。
第九方面，本申请提供了如第一方面或第二方面所述的多肽、或如第三方面所述的核酸分子、或如第四方面所述的载体、或如第五方面所述的细胞、或如第八方面所述的酶组合物在制备脱胶酶中的用途。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
实施例
提供以下实施例进一步描述本申请，而并非加以任何限制。
实验材料
本申请的实施例中所用的主要材料如下：
1.菌株
毕赤酵母SMD1168(Invitrogen，货号C17500)，大肠杆菌DH5α(TAKARA，货号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液体培养基中加入浓度1.5％的琼脂。
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液体培养基中加入浓度2％的琼脂。
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％的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(pH＝6.6)，2％琼脂，并添加10uM的ZnSO 4·7H 2O。
2％大豆磷脂乳化液的制备：称取2g大豆磷脂、100ml的H 2O，用高速匀浆机于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％的D-生物素，0.1M磷酸二氢钾-磷酸氢二钾缓冲液(pH＝6.0)。
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％的ZnSO 4·7H 2O，0.5％甲醇(灭菌后加入)，4×10 -5％的D-生物素(灭菌后加入)，0.1M的柠檬酸-柠檬酸钠缓冲液(pH＝6.6)。
3.酶活测定：pNPPC法
3.1绘制磷脂酶活力测定标准曲线
称取0.01391g对硝基苯酚，溶于50ml无菌水中，制备成2mmol/L工作液。参见表1，添加各种试剂并制作标准曲线，获得的标准曲线如图1所示。试验中测定样品酶活的条件与制作标准曲线的条件相一致。
表1：绘制磷脂酶活力测定标准曲线时的试剂添加量。
Figure PCTCN2019128973-appb-000001
Figure PCTCN2019128973-appb-000002
将以上各种溶液混合后，在37℃处理15分钟，再加入500μl的0.5N的NaOH，于410nm测定吸光度。
3.2反应缓冲液配制
0.1M的硼酸-硼酸钠缓冲液(pH＝7.6)中含有20mM pNPPC。
取600μl上述缓冲液，加入25μl的待测酶液，于37℃反应15min，再加入500μl的0.5N NaOH，终止反应，于410nm测定吸光度。
4.酶活计算
按以下公式计算酶活：
样品酶活(U/ml)＝A(410nm吸光度)×0.1935×稀释倍数×10/15
5.蛋白浓度检测试剂：
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)。
6.实验所用的酶：
限制性内切酶Sal I(购自纽英伦生物技术(北京)有限公司)；
DNA聚合酶：
Figure PCTCN2019128973-appb-000003
DNA聚合酶(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：磷脂酶C突变体文库构建及筛选
按中国发明申请CN 201510946696.1中所述的方法制备pmAO-PLC-N63DN131SN134D-Y56H载体。
以pmAO-PLC-N63DN131SN134D-Y56H载体为模板，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的文库(委托苏州泓迅生物科技股份有限公司进行文库构建)。将质粒文库转化至大肠杆菌DH5α菌株中，将所有获得的大肠杆菌克隆洗至LB液体培养基中(含100μg/ml地氨苄青霉素)，于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M314菌株的感受态细胞中。将转化物接种于MGYS平板上，于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，接种于BMM-大豆磷脂筛选平板上。选取白色沉淀圈大的克隆。获得突变体菌株，将其指定为PLC-9-49。
实施例2：磷脂酶C突变体序列分析
将PLC-9-49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PLC-9-49菌株的基因组DNA为模板，使用
Figure PCTCN2019128973-appb-000004
DNA聚合酶和引物对AOX1-5/AOX1-3进行PCR扩增，得到PLC-9-49菌株中PLC的DNA序列。其中，
引物AOX1-5的序列为：5’-GACTGGTTCCAATTGACAACG-3’；
引物AOX1-3的序列为：5’-GGCAAATGGCATTCTGACATCCTC-3’。
将获得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。PLC-9-49的DNA测序结果如SEQ ID No:3所示。经过比对发现，与SEQ ID No:1相比，SEQ ID No:3中有数个碱基发生了突变，使得SEQ ID No:2中的第6位的赖氨酸突变为脯氨酸、第8位的赖氨酸突变为异亮氨酸、第10位的甘氨酸突变为苏氨酸、第104位的赖氨酸突变为甘氨酸以及第205位的丝氨酸突变为酪氨酸。PLC-9-49的氨基酸序列如SEQ ID No:4所示。
实施例3：PLC-9-49突变体热稳定性分析
取PLC-9-49菌株及PLC-N63DN131SN134D-Y56H菌株，先在液体YPD中活化，然后接种于BMGY培养基中，于30℃以220rpm的转速振荡培养过夜。将培养物转至BMMY培养基中，初始OD 600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，于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(20mM柠檬酸-柠檬酸钠缓冲液(pH＝6.6)，含有10uM的ZnSO 4)中。
将超滤浓缩后的发酵液在60℃、65℃、70℃和75℃条件下保温 2h，取0.5μl发酵液浓缩液添加至600μl pNPPC反应缓冲液中，于37℃反应15min，再加入500μl 0.5N的NaOH终止反应，于410nm测定吸光度。根据标准曲线，计算得到各个发酵液样品的磷脂酶C活力。
PLC-9-49和PLC-N63DN131SN134D-Y56H的热稳定性如图2所示，在60℃条件下保温2h，PLC-9-49突变株的活力保留了91％，而PLC-N63DN131SN134D-Y56H的活力下降至44％。在70℃条件下保温2h，PLC-9-49突变株的活力保留了83％，而PLC-N63DN131SN134D-Y56H的活力下降至13％。在75℃条件下保温2h，PLC-9-49突变株的活力保留了61％，而PLC-N63DN131SN134D-Y56H的活力下降至3％。
由此可见，PLC-9-49的热稳定性较PLC-N63DN131SN134D-Y56H有明显的提高。
实施例4：PLC-9-49脱胶测试
取大豆毛油100g，分别加热至55℃和60℃进行脱胶，分别加入50ppm的PLC-9-49和PLC-N63DN131SN134D-Y56H样品，使得体系中的水相为3％，用高速剪切机高速(10000r/min)剪切1min，在55℃和60℃搅拌(750r/min)反应2h，升温至85℃，维持5min。将样品以12000rpm离心10min，取约10g上层油样，用HPLC检测其DAG含量。PLC-9-49样品和PLC-N63DN131SN134D-Y56H样品相对于毛油的DAG增量如图3所示。PLC-9-49在60℃脱胶时相比于在55℃脱胶时，DAG的增量增加了约10％，而PLC-N63DN131SN134D-Y56H在60℃脱胶时相较于在55℃脱胶时，DAG的增量减少了9.4％。
由此可见，PLC-9-49的脱胶温度相比于PLC-N63DN131SN134D-Y56H上升约5℃，因此工业适用性更好。

Claims (10)

  1. 具有磷脂酶C活性的多肽，所述多肽具有：
    1)包含在一个或多个位置发生氨基酸取代的SEQ ID No:2所示的氨基酸序列，其中所述一个或多个位置选自SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位或其任意组合；优选地，SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位和第205位全部发生氨基酸取代；或
    2)与1)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，且第6位、第8位、第10位、第104位、第205位的至少1个位置与SEQ ID No:2所示的氨基酸序列中第6位、第8位、第10位、第104位、第205位不同。
  2. 如权利要求1所述的多肽，其中
    SEQ ID No:2所示的氨基酸序列的第6位的氨基酸取代为脯氨酸、甘氨酸、羟脯氨酸、丝氨酸或苏氨酸取代赖氨酸，优选脯氨酸取代赖氨酸，和/或
    第8位的氨基酸取代为异亮氨酸、亮氨酸、缬氨酸、甲硫氨酸、丙氨酸、苯丙氨酸或正亮氨酸取代赖氨酸，优选异亮氨酸取代赖氨酸，和/或
    第10位的氨基酸取代为苏氨酸或丝氨酸取代甘氨酸，优选苏氨酸取代甘氨酸，和/或
    第104位的氨基酸取代为甘氨酸或脯氨酸取代赖氨酸，优选甘氨酸取代赖氨酸，和/或
    第205位的氨基酸取代为酪氨酸、色氨酸、苯丙氨酸、或苏氨酸取代丝氨酸，优选酪氨酸取代丝氨酸；
    优选地，所述多肽的氨基酸序列包含如SEQ ID No:4所示的氨基酸序列或由如SEQ ID No:4所示的氨基酸序列组成。
  3. 分离的多肽，其中，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，并且所述分离的多肽在对应于SEQ ID NO:4的第6位、第8位、第10位、第104位和第205位上的氨基酸残基分别是脯氨酸、异亮氨酸、苏氨酸、甘氨酸和酪氨酸，优选的，所述多肽获得自枯草芽孢杆菌(Bacillus subtilis)。
  4. 编码权利要求1-3中任一项所述的多肽的核酸分子，优选地，所述核酸分子包含如SEQ ID No:3所示的核酸序列或者所述核酸分子的序列如SEQ ID No:3所示。
  5. 包含权利要求4所述的核酸分子的载体，优选地，所述载体为表达载体，更优选地，所述载体被设计用于真核细胞或原核细胞中表达，尤其优选地，用于细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞中表达。
  6. 包含权利要求4所述的核酸分子或权利要求5所述的载体的细胞，优选为真核细胞或原核细胞，更优选为细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞。
  7. 使用权利要求6所述的细胞产生的磷脂酶C。
  8. 如权利要求1-3中任一项所述的多肽、或权利要求4所述的核酸分子编码的多肽、或权利要求5所述的载体编码的多肽、或权利要求6所述的细胞表达的发酵液、浓缩液或多肽、或权利要求7所述的磷脂酶C作为磷脂酶C的用途，优选为在油脂脱胶工艺中的用途。
  9. 酶组合物，其包括权利要求1-3中任一项所述的多肽或权利要求4所述的核酸分子编码的多肽、或权利要求5所述的载体编码的多 肽、或权利要求6所述的细胞表达的多肽、或权利要求7所述的磷脂酶C以及至少一种脱胶酶；优选地，所述至少一种脱胶酶选自：磷脂酶A 1、磷脂酶A 2、磷脂酶B、磷脂酶D、果胶酶和甘露聚糖酶。
  10. 如权利要求1-3中任一项所述的多肽、或如权利要求4所述的核酸分子、或如权利要求5所述的载体、或如权利要求6所述的细胞、或如权利要求9所述的酶组合物在制备脱胶酶中的用途。
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US20220073889A1 (en) 2022-03-10
EP3904510A1 (en) 2021-11-03
EP3904510A4 (en) 2022-10-19
CN111378634A (zh) 2020-07-07

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