WO2015067161A1 - 磷脂酶c突变体及其用途 - Google Patents

磷脂酶c突变体及其用途 Download PDF

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WO2015067161A1
WO2015067161A1 PCT/CN2014/090213 CN2014090213W WO2015067161A1 WO 2015067161 A1 WO2015067161 A1 WO 2015067161A1 CN 2014090213 W CN2014090213 W CN 2014090213W WO 2015067161 A1 WO2015067161 A1 WO 2015067161A1
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plc
seq
amino acid
cell
acid sequence
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PCT/CN2014/090213
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French (fr)
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许骏
宣姚吉
李金敏
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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 US15/035,105 priority Critical patent/US10144919B2/en
Publication of WO2015067161A1 publication Critical patent/WO2015067161A1/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
    • 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)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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 relates to phosphatidylcholine-specific phospholipase C mutants and uses thereof.
  • Degumming is an important step in oil refining.
  • the traditional hydration degumming method has high economic cost, high material energy consumption and heavy environmental pollution. Therefore, in recent years, many work has been devoted to degumming enzymatic degumming for degumming in oil refining. Compared with the traditional method, 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.
  • One enzyme used in the degumming of fats is a phospholipase. Phospholipase C (PLC) exhibits greater advantages than other degumming enzymes, for example, increasing the yield of glycidyl ester (DAG) and reducing the loss of oil.
  • Phospholipase C Phospholipase C
  • BC-PC-PLC Phosphatidylcholine-specific phospholipase C
  • BC-PC-PLC is 283 amino acids in length and contains a 24 amino acid signal peptide and a 14 amino acid leader peptide.
  • the mature peptide is 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 phosphatidylcholine-preferring phospholipase C of Bacillus cereus.” Gene 65(2): 293-304).
  • the crystal structure of BC-PC-PLC has been reported to consist of multiple helical domains with a catalytic site of 55 aspartic acid and 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 application provides a polypeptide having phosphatidylcholine-specific phospholipase C activity, comprising a mutated amino acid sequence set forth in SEQ ID No: 2, or an active fragment thereof, wherein the mutation comprises SEQ The asparagine at position 63 of the amino acid sequence shown by ID No: 2 was mutated.
  • the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to serine (S), alanine (A), phenylalanine (F), histidine (H) ), lysine (K), arginine (R), tryptophan (W), tyrosine (Y), cysteine (C), aspartic acid (D), glutamic acid ( E), glycine (G), isoleucine (I), leucine (L), methionine (M), glutamine (Q), threonine (T) or valine (V).
  • the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to serine (S).
  • the mutation further comprises the substitution of the arginine at position 20 of the amino acid sequence of SEQ ID No: 2 with histidine and the alanine at position 83 with aspartic acid.
  • the amino acid sequence of the polypeptide comprises a SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44 , amino acid sequence of 46 or 48 or selected from the group consisting of SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46 or 48
  • the amino acid sequence consists of.
  • the amino acid sequence of the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID No: 12.
  • the application provides a nucleic acid molecule encoding the polypeptide of the first aspect.
  • the application also provides a nucleic acid molecule comprising SEQ ID No: 7, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45 or A nucleic acid sequence of 47.
  • the application also provides a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID No:11.
  • the application provides a vector comprising the nucleic acid molecule of the second aspect.
  • the vector is an expression vector. In some embodiments, the vector is designed for expression in a eukaryotic or prokaryotic cell. In some embodiments, the vector is designed for expression in a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell.
  • the application provides a cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.
  • the cell is a eukaryotic cell or a prokaryotic cell.
  • the cell is a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell.
  • the present application provides the phospholipase C produced by the cell of the fourth aspect.
  • the present invention provides the polypeptide of the first aspect, or the polypeptide encoded by the nucleic acid molecule of the second aspect, or the vector encoded by the vector of the third aspect, or the cell expression of the fourth aspect
  • the polypeptide or the phospholipase C of the fifth aspect is used as the phosphatidylcholine-specific phospholipase C.
  • the use is in a grease degumming process.
  • the present application provides the polypeptide of the first aspect, or the nucleic acid molecule of the second aspect, or the vector of the third aspect, or the cell of the fourth aspect, for use in preparing a degumming enzyme .
  • Figure 1 is a MM- of wild-type BC-PC-PLC Pichia pastoris expressing strains G15 and 1-3 obtained in Example 1.
  • the results of the yolk selection plate showed that the top 4 clones were G15 and the lower 6 clones were 1-3, wherein the strain was cultured at 30 ° C for three days.
  • the name of the corresponding strain is indicated directly below the white sedimentation circle.
  • Fig. 2 is a graph showing the results of the MM-yolk screening panel of the mutant strain 7-3-3 obtained in Example 2, in which the strain was cultured at 30 ° C for 2 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.
  • Fig. 3 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-R20H obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.
  • Fig. 4 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-N63S obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.
  • Fig. 5 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-A83D obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.
  • Fig. 6 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-R20HN63SA83D obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.
  • Figure 7 shows the ratio of the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1 and the four BC-PC-PLC point mutation Pichia pastoris expression strains obtained in Example 4. Enzyme activity.
  • Figure 8 shows the shaking of the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1 and the four BC-PC-PLC point mutation Pichia pastoris expression strains obtained in Example 4. SDS-PAGE electropherogram of the bottle fermentation broth.
  • Figure 9 shows the shake flask fermentation broth protein concentration of the 17 BC-PC-PLC point mutation Pichia pastoris expression strains obtained in Example 6.
  • Fig. 10 is a view showing the SDS-PAGE electrophoresis of the shake flask fermentation broth of the partial BC-PC-PLC point mutation Pichia pastoris expression strain obtained in Example 6.
  • Figure 11 shows the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1, the BC-PC-PLC point mutation Pichia pastoris expression strain 6-6 obtained in Example 6 and 7-6 and the specific enzyme activities of the 18 BC-PC-PLC point mutant Pichia pastoris expressing strains obtained in Example 8 under the reaction conditions of 37 ° C and 60 ° C.
  • Figures 12 and 13 show the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1, respectively, and the BC-PC-PLC point mutation Pichia pastoris expression strain 6 obtained in Example 6 The thermal stability of the BC-PC-PLC point mutant Pichia pastoris expressing strains obtained in -6 and 7-6 and in Example 8.
  • the phosphatidylcholine phospholipase C described herein is synonymous with phosphatidylcholine-preferring phospholipase C and is also readily available to those skilled in the art. Understand.
  • a shorthand PC-PLC is used herein to mean phosphatidylcholine-specific phospholipase C or phosphatidylcholine-preferred phospholipase C.
  • An example of a phosphatidylcholine-specific phospholipase C for use herein is the phosphatidylcholine-specific phospholipase C of Bacillus cereus, denoted herein by the abbreviated BC-PC-PLC.
  • BC-PC-PLC may represent the wild-type phosphatidylcholine-specific phospholipase C of Bacillus cereus, which may also be referred to in the present application based on the wild-type phosphatidylcholine-specific phospholipase C. The mutant obtained.
  • SEQ ID No: 2 is the wild type phosphatidylcholine-specific phospholipase C of Bacillus cereus The amino acid sequence of the mature peptide.
  • polypeptide peptide
  • protein protein
  • nucleic acid and “polynucleotide” as used herein are used interchangeably and include, but are 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.
  • the present application provides phospholipase C mutants obtained by mutation screening methods in molecular biology and uses thereof.
  • the phospholipase C may be phosphatidylcholine-specific phospholipase C (PC-PLC). More specifically, the phosphatidylcholine-specific phospholipase C may be phosphatidylcholine-specific phospholipase C (BC-PC-PLC) of Bacillus cereus.
  • PC-PLC phosphatidylcholine-specific phospholipase C
  • BC-PC-PLC phosphatidylcholine-specific phospholipase C
  • the application provides a polypeptide having phosphatidylcholine-specific phospholipase C activity, comprising a mutated amino acid sequence set forth in SEQ ID No: 2, or an active fragment thereof, wherein the mutation comprises SEQ The asparagine at position 63 of the amino acid sequence shown by ID No: 2 was mutated.
  • the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to alanine (A), cysteine (C), aspartic acid (D), glutamine Acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M) ), glutamine (Q), arginine (R), threonine (T), valine (V), tryptophan (W) or tyrosine (Y).
  • the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to serine (S).
  • the mutation further comprises the substitution of the arginine at position 20 of the amino acid sequence of SEQ ID No: 2 with histidine and the alanine at position 83 with aspartic acid.
  • the amino acid sequence of the polypeptide comprises a SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44 , amino acid sequence of 46 or 48 or selected from the group consisting of SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46 or 48
  • the amino acid sequence consists of.
  • the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44
  • the amino acid sequence of 46 or 48 that is, the amino acid sequence shown by SEQ ID No: 2 differs only in the amino acid mutation at position 63 described above.
  • the amino acid sequence of the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID No: 12. In some embodiments, the amino acid sequence of the polypeptide consists of the amino acid sequence set forth in SEQ ID No: 12, ie, differs from the amino acid sequence set forth in SEQ ID No: 2 only in the 20th position described above.
  • the mutation was histidine (H), the mutation at position 63 was serine (S), and the mutation at position 83 was aspartic acid (D).
  • amino acid sequence of the polypeptide is the same as the length of the amino acid sequence set forth in SEQ ID No: 2.
  • the amino acid sequence of the polypeptide has a length greater than the amino acid sequence set forth in SEQ ID No: 2.
  • the polypeptide further comprises a signal peptide and/or a leader peptide.
  • the wild type phosphatidylcholine-specific phospholipase C of Bacillus cereus comprises a 24 amino acid signal peptide and a 14 amino acid leader peptide, and thus the polypeptide of the present application may also comprise the same Or other signal peptides and/or leader peptides.
  • the signal peptide is an alpha factor signal peptide.
  • polypeptides of the present application may also include other functional elements such as, but not limited to, tag elements for isolation and purification (eg, histidine tags), selection elements (eg, based on antibiotic selection or fluorescent selection) (eg green fluorescent protein, GFP)) and the like.
  • tag elements for isolation and purification eg, histidine tags
  • selection elements eg, based on antibiotic selection or fluorescent selection
  • green fluorescent protein, GFP green fluorescent protein
  • the polypeptide has an amino acid sequence that is less than the amino acid sequence set forth in SEQ ID No: 2.
  • the polypeptide may comprise a mutated active fragment of the amino acid sequence set forth in SEQ ID No: 2, such as SEQ ID Nos: 8, 12, 14, 16, 18, 20, 22, 24, 26, An active fragment of the amino acid sequence shown at 28, 30, 32, 34, 38, 40, 42, 44, 46 or 48.
  • active fragment denotes a part of a wild-type phosphatidylcholine-specific phospholipase C or a phosphatidylcholine-specific phospholipase C mutant of the present application which still retains phosphatidylcholine-specific phospholipids.
  • the 8th and 9th alpha helices are amino acids 140-153 and 154-157
  • the amino acid, the 10th alpha helix is amino acids 171-186, so the predicted active fragment is 1-170 amino acids.
  • the present application also contemplates functional variants of the polypeptides described in the first aspect.
  • the functional variant is a conservative substitution variant.
  • Constant substitution refers to a change in the amino acid composition of a protein that does not significantly alter the activity of the protein.
  • “conservative substitution” of a particular amino acid sequence refers to the substitution of those amino acids that are not critical to protein activity, or with similar properties (eg, acidic, basic, positively or negatively charged, polar or non-polar, etc.)
  • the other amino acids replace the amino acids so that even the substitution of the key amino acids does not significantly alter the activity.
  • Conservative substitution representatives that provide functionally similar amino acids are well known in the art. For example, each of the 6 groups in the table below includes amino acids that are conservatively substituted with each other:
  • the functional variant and the parent sequence eg, SEQ ID No: 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40
  • the amino acid sequence of 42, 42, 44 or 48 has an identity or similarity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher.
  • the application provides a nucleic acid molecule encoding the polypeptide of the first aspect.
  • the present application contemplates different nucleic acid molecules that are obtainable due to the degeneracy of the genetic code or the preference of different species for codons.
  • the application also provides a nucleic acid molecule comprising SEQ ID No: 7, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45 or 47 nucleic acid sequence.
  • the application also provides a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID No:11.
  • the nucleic acid molecules of the present application may comprise not only the coding sequences of BC-PC-PLC mature peptide mutants, but also other nucleic acid sequences.
  • the additional nucleic acid sequence is a nucleic acid sequence encoding a signal peptide and/or a leader peptide.
  • the additional nucleic acid sequence is a nucleic acid sequence encoding a tagging element (eg, a histidine tag) for isolation and purification, encoding a selection element (eg, based on antibiotic selection or fluorescent selection (eg, green fluorescent protein, GFP) ))
  • the nucleic acid sequence eg, based on antibiotic selection or fluorescent selection (eg, green fluorescent protein, GFP)
  • the other nucleic acid sequences may also be regulatory sequences required for transcription and/or translation, such as promoters, enhancers, and the like.
  • the application provides a vector comprising the nucleic acid molecule of the second aspect.
  • the vector is an expression vector.
  • the vector is designed for use in a eukaryotic or prokaryotic cell expression.
  • the vector is designed for expression in a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell.
  • the vector is a plasmid. Suitable eukaryotic or prokaryotic vectors are well known to those skilled in the art, and a variety of parent carriers are commercially available. Examples of vectors include, but are not limited to, a variety of vectors used in embodiments of the present application.
  • the application provides a cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.
  • the cell is a eukaryotic cell or a prokaryotic cell.
  • the cell is a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell.
  • the cell is a pichia pastoris cell.
  • the cell is a Bacillus subtilis cell.
  • the nucleic acid molecule can be located extrachromosomally (e.g., in a vector) or can be integrated into the chromosome of a host cell.
  • Techniques for integrating a nucleic acid molecule into the chromosome of a 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 the phospholipase C produced by the cell of the fourth aspect.
  • Techniques for producing a polypeptide or protein of interest using genetically engineered host cells are well known to those skilled in the art.
  • the present invention provides the polypeptide of the first aspect, or the polypeptide encoded by the nucleic acid molecule of the second aspect, or the vector encoded by the vector of the third aspect, or the cell expression of the fourth aspect
  • the polypeptide or the phospholipase C of the fifth aspect is used as the phosphatidylcholine-specific phospholipase C.
  • the use is in a grease degumming process.
  • the use of phosphatidylcholine-specific phospholipase C in a grease degumming process is known in the art.
  • Phospholipase C is capable of hydrolyzing the colloidal phospholipids in the oil to form a hydrophilic phosphate moiety and a lipophilic DAG.
  • the hydrophilic portion is carried away by water to remove the colloidal fraction, and DAG increases the oil yield.
  • the feedstock oil is heated to 60 ° C, the phospholipase C solution is added, and after high-speed shear mixing, the reaction is stirred in the reactor for 2 h, and then the aqueous phase and the oil phase are separated by centrifugation.
  • the present application provides the polypeptide of the first aspect, or the nucleic acid molecule of the second aspect, or the vector of the third aspect, or the cell of the fourth aspect, for use in preparing a degumming enzyme .
  • Methods for preparing degumming enzymes using polypeptides, nucleic acid molecules, vectors or transformed cells are well known in the art.
  • the DNA sequence of the isolated phospholipase C sequence can be transformed into a host cell (for example, Pichia pastoris) by an expression vector, and subjected to large-scale fermentation culture in a fermenter, and the fermentation liquid is obtained by filtration, and then passed through ultrafiltration.
  • the corresponding buffer is replaced to remove the higher concentration of salt ions in the fermentation broth, and a common stabilizer (such as glycerin) and metal ion zinc (added as zinc sulfate) are added to the ultrafiltrate.
  • Pichia pastoris GS115 Invitrogen, Cat. No. C181-00
  • Escherichia coli DH5a TAKARA, Cat. No. D9057A
  • Plasmid pPIC-9k (Invitrogen, Cat. No. V17520), pAO815 plasmid (Invitrogen, Cat. No. V18020), pAO-PLC plasmid and pAOmu-PLC plasmid were constructed by the inventors of the present application, as described in detail below.
  • LB liquid medium 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0.
  • LB solid medium 1.5% agar was added to the LB liquid medium.
  • YPD liquid medium 1% yeast extract, 2% peptone, 2% glucose.
  • YPD solid medium 2% agar was added to the LB liquid medium.
  • MGYS solid medium 1.34% yeast nitrogen source base (YNB) (containing ammonium sulfate, no amino acid), 1% glycerol, 1 M sorbitol, 4 x 10 -5 % D-biotin, 2% agar.
  • YNB yeast nitrogen source base
  • MM-yolk screening medium 1.34% yeast nitrogen source base (YNB) (containing ammonium sulfate, no amino acid), 4 ⁇ 10 -5 % D-biotin, 0.5% methanol (added after sterilization), 2% egg yolk Liquid, 2% agar.
  • yeast nitrogen source base YNB
  • YNB yeast nitrogen source base
  • Preparation of egg yolk solution Take one fresh egg, wipe it with 75% alcohol, knock the eggshell with sterile scorpion to make the egg white flow out, rinse the egg yolk with sterile water twice, and add it to a triangular bottle containing 80ml of sterile water. Mix well to obtain 20% egg yolk.
  • BMGY liquid medium 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) (ammonium sulfate, no amino acid), 1% glycerol, 4 ⁇ 10 -5 % D-biotin, 0.1 M potassium dihydrogen phosphate-dipotassium hydrogen 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) (ammonium sulfate, no amino acid), 0.5% methanol (added after sterilization), 4 ⁇ 10 -5 % D-biotin (added after sterilization), 0.1 M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 6.0).
  • yeast nitrogen source base YNB
  • PLC reaction solution 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl 2
  • CIAP reaction solution 50 mM Tris-HCl pH 9.0, 10 mM MgCl 2 , 1 U CIAP (purchased from Bao Bioengineering (Dalian) Co., Ltd.)
  • Molybdenum blue color reaction solution 100 ⁇ l CIAP reactant, 0.2% ascorbic acid, 0.1% ammonium molybdate (prepared with 30% H 2 SO 4 )
  • PCR enzyme TaKaRa Taq, DNA polymerase (purchased from Bao Bioengineering (Dalian) Co., Ltd.)
  • T4DNA ligase (purchased from Enzyme Co., Ltd.)
  • BC-PC-PLC was designed according to the mature peptide sequence of phosphatidylcholine-specific phospholipase C of Bacillus cereus (PDB ID: 1AH7) and Pichia codon preference (SEQ ID No: 1). And fused the alpha factor signal peptide sequence at its front end (the DNA sequence of which is derived from the commercial Pichia pastoris expression vector pPIC-9k, positions 8-274 of SEQ ID No: 3) and the Kozak sequence of Pichia pastoris (SEQ. ID No: 3, 1st to 7th), the ⁇ -BC-PC-PLC DNA sequence (SEQ ID No: 3) was finally obtained.
  • the ⁇ -BC-PC-PLC DNA sequence was supplied to Shanghai Shenggong Biotechnology Co., Ltd. for whole gene synthesis, and the cloning vector pGEM-T-PLC containing the ⁇ -BC-PC-PLC DNA sequence was obtained. Use this carrier as a template, use The DNA polymerase and the primer pair AmPLC-3/AmPLC-4 were amplified by PCR to obtain a PLC fragment.
  • the PAOX1+PLC fusion fragment was cloned into the pAO815 vector using AatII and EcoRI cleavage sites to obtain the expression vector pAO-PLC.
  • the pAO-PLC was linearized with SalI and the gel was recovered to an approximately 8.5 kb fragment.
  • Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and the linearized pAO-PLC fragment was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked and suspended in 5 ⁇ l of sterile water.
  • 0.5 ⁇ l was inoculated on the MM-yolk screening plate. After culturing at 30 ° C for 3 days, a positive clone of a white sediment circle was observed around the cells.
  • the expressed phosphatase C can decompose lecithin into phosphatidylcholine and water-insoluble diglyceride, and the white sinking ring around the colony of MM-yolk screening plate belongs to the above reaction.
  • the white precipitate circle formed around the colony is relatively large.
  • Fig. 1 Two positive strains were screened, as shown in Fig. 1, which were designated as G15 and 1-3, respectively.
  • the white precipitate circle of G15 was smaller, and the white precipitate circle of 1-3 was larger.
  • pAO-PLC vector as template DNA polymerase and primer pair AmPLC-1/AOXH-2 were amplified by PCR to obtain a fragment of about 900 bp.
  • pAO-PLC vector as template DNA polymerase and primer pair AOXH-3/AmPLC-4, amplified by PCR to obtain a fragment of about 1.1 kb.
  • the approximately 900 bp fragment obtained by the previous two-step PCR and the about 1.1 kb fragment were mixed as a template for the third step PCR, using primer pair AmPLC. -1/AmPLC-4 and DNA polymerase was amplified by PCR to obtain a fragment of about 1.9 kb.
  • the approximately 1.9 kb fragment was cloned into pAO-PLC by AatII and EcoRI cleavage sites to obtain pmAO-PLC.
  • pmAO-PLC a HindIII restriction site in pAO-PLC was mutated to retain only one HindIII restriction site located at the 5' end of the BC-PC-PLC sequence, enabling the use of HindIII and EcoRI for BC
  • the mutated fragment of the -PC-PLC was cloned into pmAO-PLC.
  • the pAO-PLC vector was used as a template, and the error-prone PCR was performed on EPPLC-1/EPPLC-2 using TaKaRa Taq enzyme and primers (addition of 0.3 mM MnCl 2 at the time of PCR) to obtain a mutant amplicon fragment of about 755 bp in size. set.
  • the obtained fragment was cloned into pmAO-PLC by HindIII and EcoRI digestion sites, and the resulting vector was transformed into Escherichia coli DH5 ⁇ strain, and a total of 1 ⁇ 10 4 BC-PC-PLC mutants were obtained.
  • Each 1 ⁇ 10 3 BC-PC-PLC mutants were washed with 2 ml of sterile water into 8 ml of 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.
  • 500 ng of vector (using as little DNA as possible to ensure that most positive transformants contain a single copy of the PLC gene) was transformed into competent cells of the Pichia pastoris GS115 strain by electroporation.
  • the transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days to obtain a Pichia pastoris mutant library of BC-PC-PLC.
  • the monoclonal on the plate was picked, suspended in 5 ⁇ l of sterile water, and 0.5 ⁇ l was inoculated on the MM-yolk screening plate.
  • the size of the white precipitate circle in the mutant and G15 was compared with G15 as a control.
  • a final mutant strain with a white precipitated ring larger than G15 was designated for screening, designated 7-3-3, as shown in Figure 2.
  • the 7-3-3 strain was inoculated into 3 ml of YPD liquid medium, cultured at 30 ° C overnight, and genomic DNA was extracted. Using the genomic DNA of 7-3-3 strain as a template, use The DNA polymerase and the primers were subjected to PCR amplification of AOX1-5/AOX1-3 to obtain the DNA sequence of BC-PC-PLC in the 7-3-3 strain. The obtained sequence was sent to Shanghai Shenggong Bioengineering Co., Ltd., and the primers were used to sequence AOX1-5/AOX1-3. The DNA sequencing results of 7-3-3 BC-PC-PLC are shown in SEQ ID No: 4.
  • pAO-PLC vector as template DNA polymerase and primer pair EPPLC-1/20RH-2 were amplified by PCR to obtain a fragment of about 78 bp.
  • pAO-PLC vector as template DNA polymerase and primer pair 20RH-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 707 bp.
  • the approximately 78 bp fragment obtained by the previous two-step PCR and the approximately 707 bp fragment were mixed as a template for the third step PCR, using the primer pair EPPLC-1/EPPLC-2 and DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.
  • the about 755 bp fragment was cloned into pmAO-PLC by HindIII and EcoRI cleavage sites to obtain a pmAO-PLC-R20H vector.
  • the pmAO-PLC-R20H was linearized with SalI, and the 8.5 kb fragment was recovered by gel.
  • Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-R20H was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days.
  • the monoclonal on the plate was picked, suspended in 5 ⁇ l of sterile water, and 0.5 ⁇ l was inoculated on the MM-yolk screening plate.
  • the size of the mutant and the G15 and 1-3 white sedimentary circles were compared with G15 and 1-3 as shown in Fig. 3.
  • a negative clone was found on the selection plate without a white sedimentary circle, ie, the PLC gene fragment was not successfully transferred into the genome of the clone.
  • the mutant clones thus selected often contain a single copy of the PLC gene, which facilitates reducing the effect of differences in expression levels on enzyme activity comparison.
  • pAO-PLC vector as template DNA polymerase and primer pair EPPLC-1/63NS-2, amplified by PCR to obtain a fragment of about 207 bp.
  • pAO-PLC vector as template DNA polymerase and primer pair 63NS-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 576 bp.
  • the 207 bp fragment and the approximately 576 bp fragment obtained by the previous two-step PCR were mixed as a template for the third step PCR, using the primer pair EPPLC-1/EPPLC-2 and DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.
  • the about 755 bp fragment was cloned into pmAO-PLC by HindIII and EcoRI cleavage sites to obtain pmAO-PLC-N63S vector, pmAO-PLC-N63S was linearized with SalI, and the 8.5 kb fragment was recovered by gel.
  • Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-N63S was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days.
  • the monoclonal on the plate was picked, suspended in 5 ⁇ l of sterile water, and 0.5 ⁇ l was inoculated on the MM-yolk screening plate.
  • the size of the mutant and the G15 and 1-3 white sedimentation circles were compared with G15 and 1-3 as shown in Fig. 4.
  • the mutant positive clone PLC-N63S was used for subsequent experiments as described in Example 4.1.
  • pAO-PLC vector as template DNA polymerase and primer pair EPPLC-1/83AD-2 were amplified by PCR to obtain a fragment of about 266 bp.
  • pAO-PLC vector as template DNA polymerase and primer pair 83AD-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 520 bp.
  • the 266 bp fragment and the approximately 502 bp fragment obtained by the previous two-step PCR were mixed as a template for the third step PCR, using primer pairs EPPLC-1/EPPLC-2 and DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.
  • the about 755 bp fragment was cloned into pmAO-PLC by HindIII and EcoRI cleavage sites to obtain pmAO-PLC-A83S vector.
  • the pmAO-PLC-A83S was linearized with SalI, and the 8.5 kb fragment was recovered by gel.
  • Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-A83D was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days.
  • the monoclonal on the plate was picked, suspended in 5 ⁇ l of sterile water, and 0.5 ⁇ l was inoculated on the MM-yolk screening plate.
  • the size of the mutant and the G15 and 1-3 white sedimentary circles were compared with G15 and 1-3 as shown in Fig. 5.
  • the mutant positive clone PLC-A83D was used for subsequent experiments as described in Example 4.1.
  • pmAO-PLC-R20HN63SA83D This fragment was cloned into pmAO-PLC by HindIII and EcoRI to obtain a pmAO-PLC-R20HN63SA83D vector.
  • the pmAO-PLC-R20HN63SA83D was linearized with SalI, and the 8.5 kb fragment was recovered by gel.
  • Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-R20HN63SA83D was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days.
  • the monoclonal on the plate was picked, suspended in 5 ⁇ l of sterile water, and 0.5 ⁇ l was inoculated on the MM-yolk screening plate.
  • the size of the mutant and the G15 and 1-3 white precipitation circles were compared with G15 and 1-3 as shown in Fig. 6.
  • the mutant positive clone PLC-R20HN63SA83D was used for subsequent experiments as described in Example 4.1.
  • the white precipitate on the screening plate of the PLC-N63S mutant was substantially equivalent to the white precipitate of the PLC-R20HN63SA83D clone and was significantly larger than the white precipitates of G15 and 1-3.
  • the size of the PLC-R20H and PLC-A83D mutants on the white precipitation circle is approximately the same as the white precipitation circle size of G15 and 1-3.
  • 10 ⁇ l of the fermentation broth was added to 190 ⁇ l of PLC reaction solution (containing 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl 2 ), and incubated at 37 ° C for 30 min with shaking. After the incubation, 100 ⁇ l of chloroform was added and shaken, and centrifuged at 12,000 rpm for 2 min. 80 ⁇ l of the supernatant was taken, and 20 ⁇ l of CIAP reaction solution (containing 50 mM Tris-HCl pH 9.0, 10 mM MgCl 2 , 1 U CIAP) was added, and the mixture was incubated at 37 ° C for 1 h with shaking.
  • PLC reaction solution containing 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl 2
  • CIAP reaction solution containing 50 mM Tris-HCl pH 9.0, 10 mM MgCl 2 , 1 U CIA
  • the protein concentration of the shake flask fermentation broth of G15, 1-3, PLC-R20H, PLC-N63S, PLC-A83D and PLC-R20HN63SA83D strains was determined by Bradford reagent, and the results were as follows: G15, 1-3, PLC-R20H, PLC -N63S, PLC-A83D and PLC-R20HN63SA83D specific enzyme activity. As shown in Fig.
  • the specific enzyme activity of PLC-N63S and PLC-R20HN63SA83D is about 4 times that of wild type, and the specific enzyme activity of PLC-R20H and PLC-A83D is equivalent to wild type, which proves that the 63th asparagine mutation is Serine is a key mutation site that is increased in activity over enzyme activity.
  • the protein amount of the shake flask fermentation broth of G15, 1-3, PLC-R20H, PLC-N63S, PLC-A83D and PLC-R20HN63SA83D strains was adjusted to the same amount for SDS-PAGE electrophoresis, and the results are shown in Fig. 8.
  • the PLC-N63S PLC protein band is slightly less intense than the G15 PLC protein band, so the specific enzyme activity of PLC-N63S is at least 4 times that of G15.
  • Example 6 Construction of high-yield strains of PLC-N63S and PLC-R20HN63SA83D and shake flask fermentation
  • pmAO-PLC-N63S and pmAO-PLC-R20HN63SA83D were linearized with SalI, and the gel recovered approximately 8.5 kb fragment.
  • Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 2 ⁇ g of linearized pmAO-PLC-R20HN63SA83D was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked, suspended in 5 ⁇ l of sterile water, and 0.5 ⁇ l was inoculated on the MM-yolk screening plate.
  • strains 6-1 to 6-11 and 7-1 to 7-6 were firstly activated in liquid YPD, then inoculated into BMGY medium, and cultured overnight at 30 ° C with shaking at 220 rpm. The culture was transferred to BMMY medium, the initial OD 600 of 6.
  • the highest concentration of fermentation broth protein was 6-6 strain, the fermentation broth protein concentration was 0.108 mg/ml, and the highest concentration of fermentation broth protein in 7-1 to 7-6 was 7- 6 strain, the fermentation broth protein concentration was 0.238 mg / ml.
  • SDS-PAGE electrophoresis was performed on several strains with higher protein concentrations in the two strains, and the amount of band protein in the PLC was compared. The results are shown in Fig. 10.
  • the intensity of the PLC target bands of 7-5 and 7-6 is higher than 6
  • the R20H and A83D mutations in the PLC-R20HN63SA83D mutant help to increase the expression level of the mutant in Pichia pastoris.
  • Example 7 amino acid saturation mutation at position 63 of BC-PC-PLC
  • pAO-PLC vector as a template DNA polymerase and primer pair EPPLC-1/63X-2 (X represents a single-letter abbreviation for the above 18 amino acids), and approximately 207 bp fragment was amplified by PCR.
  • pAO-PLC vector as template DNA polymerase and primer pair 63X-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 576 bp.
  • the 207 bp fragment and the approximately 576 bp fragment obtained by the previous two-step PCR were mixed as a template for the third step PCR, using the primer pair EPPLC-1/EPPLC-2 and DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.
  • the 18 755 bp fragments obtained were cloned into pmAO-PLC by HindIII and EcoRI cleavage sites, respectively, to obtain 18 pmAO-PLC-N63X vectors, and 18 pmAO-PLC-N63X vectors were linearized with SalI. The 8.5 kb fragment was recovered.
  • Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and then 500 ng linearized 18 pmAO-PLC-N63X were transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days.
  • the monoclonal on the plate was picked, suspended in 5 ⁇ l of sterile water, and 0.5 ⁇ l was inoculated on the MM-yolk screening plate.
  • the size of the mutant and the G15 and 1-3 white precipitation circles were compared using G15 and 1-3 as controls.
  • the mutant positive clone PLC-N63X was used for subsequent experiments as described in Example 4.1. Among them, PLC-N63P, a mutant in which the 63th amino acid was mutated to proline, was formed without a white precipitate, indicating that the mutation inactivated the PLC.
  • Example 8 Shake flask fermentation enzyme activity and thermal stability of BC-PC-PLC saturated mutant strain
  • 10 ⁇ l of the fermentation broth was added to 190 ⁇ l of PLC reaction solution (containing 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl 2 ), and incubated at 37 ° C or 60 ° C for 30 min with shaking. After the incubation, 100 ⁇ l of chloroform was added and shaken, and centrifuged at 12,000 rpm for 2 min. 80 ⁇ l of the supernatant was taken, and 20 ⁇ l of CIAP reaction solution (containing 50 mM Tris-HCl pH 9.0, 10 mM MgCl 2 , 1 U CIAP) was added, and the mixture was incubated at 37 ° C for 1 h with shaking.
  • PLC reaction solution containing 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl 2
  • CIAP reaction solution containing 50 mM Tris-HCl pH 9.0, 10 mM MgCl 2
  • PLC-N63P When reacted at 60 °C, PLC-N63P had almost no activity. PLC-N63D and PLC-N63I had no significant effect on enzyme activity improvement. The other enzymes had more than one-fold increase in enzyme activity than wild type, among which PLC-N63S, The specific enzyme activities of PLC-N63A, PLC-N63F, PLC-N63H, PLC-N63K, PLC-N63R, PLC-N63W and PLC-N63Y were more than 6 times that of wild type.
  • the specific activity of the wild type at 60 ° C was 1.5 times that of the reaction at 37 ° C, while the mutants PLC-N63S, PLC-N63A, PLC-N63C, PLC-N63D, PLC-N63E, PLC-N63F, PLC-
  • the specific enzyme activity of N63G, PLC-N63H, PLC-N63I, PLC-N63K, PLC-N63L, PLC-N63M, PLC-N63R, PLC-N63V, PLC-N63W and PLC-N63Y at 60 °C is 37 °C. 2 times or more at the time of reaction.
  • the reaction was carried out at 37 ° C and 60 ° C, respectively, to determine the specific enzyme activity after treatment at 90 ° C.
  • the mutants PLC-N63F, PLC-N63W and PLC-N63Y retained 50% viability at 37 °C after treatment at 90 °C for 1 h, while the ratio of wild type to other mutants Enzyme activity is greatly reduced.
  • the mutants PLC-N63F, PLC-N63W and PLC-N63Y remained viable at 30 °C after treatment at 90 °C for 1 h, while the wild type and the remaining mutants remained It is much lower than the enzyme activity. Description PLC-N63F, PLC-N63W and PLC-N63Y have good thermal stability.
  • the 63rd amino acid of BC-PC-PLC was mutated from asparagine to alanine (A), phenylalanine (F), glycine (G), histidine (H), and Leucine (I), lysine (K), leucine (L), methionine (M), glutamine (Q), arginine (R), threonine (T), After serine (S), tyrosine (V), tryptophan (W) and tyrosine (Y), the specific enzyme activity at 37 ° C and 60 ° C was more than 2 times higher than that of the wild type.
  • SEQ ID No: 1 wild type BC-PC-PLC DNA coding sequence
  • SEQ ID No: 2 wild type BC-PC-PLC amino acid sequence
  • SEQ ID No: 3 artificially synthesized a-BC-PC-PLC DNA sequence
  • SEQ ID No: 4 BC-PC-PLC mutant 7-3-3 DNA coding sequence
  • SEQ ID No: 5 mutant PLC-R20H DNA coding sequence
  • SEQ ID No: 6 mutant PLC-R20H amino acid sequence
  • SEQ ID No: 7 mutant PLC-N63S DNA coding sequence
  • SEQ ID No: 8 mutant PLC-N63S amino acid sequence
  • SEQ ID No: 9 mutant PLC-A83D DNA coding sequence
  • SEQ ID No: 10 mutant PLC-A83D amino acid sequence
  • SEQ ID No: 11 PLC-R20HN63SA83D DNA coding sequence
  • SEQ ID No: 12 Amino acid sequence of PLC-R20HN63SA83D;
  • SEQ ID No: 13 PLC-N63A DNA coding sequence
  • SEQ ID No: 14 PLC-N63A amino acid sequence
  • SEQ ID No: 15 PLC-N63C DNA coding sequence
  • SEQ ID No: 16 PLC-N63C amino acid sequence
  • SEQ ID No: 17 PLC-N63D DNA coding sequence
  • SEQ ID No: 18 PLC-N63D amino acid sequence
  • SEQ ID No: 19 PLC-N63E DNA coding sequence
  • SEQ ID No: 20 PLC-N63E amino acid sequence
  • SEQ ID No: 21 PLC-N63F DNA coding sequence
  • SEQ ID No: 22 PLC-N63F amino acid sequence
  • SEQ ID No: 23 PLC-N63G DNA coding sequence
  • SEQ ID No: 24 PLC-N63G amino acid sequence
  • SEQ ID No: 25 PLC-N63H DNA coding sequence
  • SEQ ID No: 26 PLC-N63H amino acid sequence
  • SEQ ID No: 27 PLC-N63I DNA coding sequence
  • SEQ ID No: 28 PLC-N63I amino acid sequence
  • SEQ ID No: 29 PLC-N63K DNA coding sequence
  • SEQ ID No: 30 PLC-N63K amino acid sequence
  • SEQ ID No: 31 PLC-N63L DNA coding sequence
  • SEQ ID No: 32 PLC-N63L amino acid sequence
  • SEQ ID No: 33 PLC-N63M DNA coding sequence
  • SEQ ID No: 34 PLC-N63M amino acid sequence
  • SEQ ID No: 35 PLC-N63P DNA coding sequence
  • SEQ ID No: 36 PLC-N63P amino acid sequence
  • SEQ ID No: 37 PLC-N63Q DNA coding sequence
  • SEQ ID No: 38 PLC-N63Q amino acid sequence
  • SEQ ID No: 39 PLC-N63R DNA coding sequence
  • SEQ ID No: 40 PLC-N63R amino acid sequence
  • SEQ ID No: 41 PLC-N63T DNA coding sequence
  • SEQ ID No: 42 PLC-N63T amino acid sequence
  • SEQ ID No: 43 PLC-N63V DNA coding sequence
  • SEQ ID No: 44 PLC-N63V amino acid sequence
  • SEQ ID No: 45 PLC-N63W DNA coding sequence
  • SEQ ID No: 46 PLC-N63W amino acid sequence
  • SEQ ID No: 47 PLC-N63Y DNA coding sequence
  • SEQ ID No: 48 PLC-N63Y amino acid sequence.

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Abstract

本申请提供了蜡样芽胞杆菌的野生型磷脂酰胆碱特异性磷脂酶C的突变体，涉及的突变包括第63位的天冬酰胺突变为其他氨基酸，还可以包括第20位的精氨酸至组氨酸以及第83位的丙氨酸至天冬氨酸的突变。本申请还提供了编码所述突变体的核酸分子、包含所述核酸分子的载体以及包含所述核酸分子或载体的细胞。本申请还提供了所述突变体、核酸分子载体以及细胞的用途。

Description

磷脂酶C突变体及其用途 发明领域
本申请涉及磷脂酰胆碱特异性磷脂酶C突变体及其用途。
发明背景
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."Gene65(2):293-304)。
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Zn2+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)。
本领域中需要改进的(例如酶活性更高的)磷脂酶C。
发明概述
第一方面，本申请提供了具有磷脂酰胆碱特异性磷脂酶C活性的多肽，所述多肽包含突变的SEQ ID No:2所示的氨基酸序列或其活性片段，其中所述突变包括将SEQ ID No:2所示的氨基酸序列的第63位的天冬酰胺进行突变。
在一些实施方案中，将SEQ ID No:2的氨基酸序列的第63位的天冬酰胺突变为丝氨酸(S)、丙氨酸(A)、苯丙氨酸(F)、组氨酸(H)、赖氨酸(K)、精氨酸(R)、色氨酸(W)、酪氨酸(Y)、半胱氨酸(C)、天冬氨酸(D)、谷氨酸(E)、甘氨酸(G)、异亮氨酸(I)、亮氨酸(L)、蛋氨酸(M)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)。在一些实施方案中，将SEQ ID No:2的氨基酸序列的第63位的天冬酰胺突变为丝氨酸(S)。
在一些实施方案中，所述突变还包括SEQ ID No:2氨基酸序列的第20位的精氨酸被组氨酸取代以及第83位的丙氨酸被天冬氨酸取代。
在一些实施方案中，所述多肽的氨基酸序列包含选自SEQ ID No:8、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48的氨基酸序列或由选自SEQ ID No:8、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48的氨基酸序列组成。
在一些实施方案中，所述多肽的氨基酸序列包含SEQ ID No:12所示的氨基酸序列或由SEQ ID No:12所示的氨基酸序列组成。
第二方面，本申请提供了编码第一方面中所述的多肽的核酸分子。
本申请还提供了核酸分子，其包含选自SEQ ID No:7、13、15、17、19、21、23、25、27、29、31、33、37、39、41、43、45或47的核酸序列。
本申请还提供了核酸分子，其包含SEQ ID No:11所示的核酸序列。
第三方面，本申请提供了包含第二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。
第四方面，本申请提供了包含第二方面所述的核酸分子或第三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。
第五方面，本申请提供了第四方面所述的细胞产生的磷脂酶C。
第六方面，本申请提供了第一方面所述的多肽、或第二方面所述的核酸分子编码的多肽、或第三方面所述的载体编码的多肽、或第四方面所述的细胞表达出的多肽、或第五方面所述的磷脂酶C作为磷脂酰胆碱特异性磷脂酶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。
第七方面，本申请提供了第一方面所述的多肽、或第二方面所述的核酸分子、或第三方面所述的载体、或第四方面所述的细胞在制备脱胶酶中的用途。
附图简要说明
图1为实施例1中获得的野生型BC-PC-PLC毕赤酵母表达菌株G15和1-3的MM- 卵黄筛选平板的结果图，上方4个克隆为G15，下方6个克隆为1-3，其中菌株在30℃下培养三天。白色沉淀圈的正下方标出了对应的菌株的名称。
图2为实施例2中获得的突变株7-3-3的MM-卵黄筛选平板的结果图，其中菌株在30℃下培养2天。白色沉淀圈的正下方标出了对应的菌株的名称。
图3为实施例4中获得的BC-PC-PLC点突变毕赤酵母表达菌株PLC-R20H的MM-卵黄筛选平板的结果图，其中菌株在30℃下培养3天。白色沉淀圈的正下方标出了对应的菌株的名称
图4为实施例4中获得的BC-PC-PLC点突变毕赤酵母表达菌株PLC-N63S的MM-卵黄筛选平板的结果图，其中菌株在30℃下培养3天。白色沉淀圈的正下方标出了对应的菌株的名称。
图5为实施例4中获得的BC-PC-PLC点突变毕赤酵母表达菌株PLC-A83D的MM-卵黄筛选平板的结果图，其中菌株在30℃下培养3天。白色沉淀圈的正下方标出了对应的菌株的名称。
图6为实施例4中获得的BC-PC-PLC点突变毕赤酵母表达菌株PLC-R20HN63SA83D的MM-卵黄筛选平板的结果图，其中菌株在30℃下培养3天。白色沉淀圈的正下方标出了对应的菌株的名称。
图7显示了实施例1中获得的野生型BC-PC-PLC毕赤酵母表达菌株G15和1-3以及实施例4中获得的4个BC-PC-PLC点突变毕赤酵母表达菌株的比酶活。
图8显示了实施例1中获得的野生型BC-PC-PLC毕赤酵母表达菌株G15和1-3以及实施例4中获得的4个BC-PC-PLC点突变毕赤酵母表达菌株的摇瓶发酵液的SDS-PAGE电泳图。
图9显示了实施例6中获得的17个BC-PC-PLC点突变毕赤酵母表达菌株的摇瓶发酵液蛋白浓度。
图10显示了实施例6中获得的部分BC-PC-PLC点突变毕赤酵母表达菌株的摇瓶发酵液SDS-PAGE电泳图。
图11显示了实施例1中获得的野生型BC-PC-PLC毕赤酵母表达菌株G15和1-3，实施例6中获得的BC-PC-PLC点突变毕赤酵母表达菌株6-6和7-6以及实施例8中获得的18个BC-PC-PLC点突变毕赤酵母表达菌株在37℃和60℃反应条件下的比酶活。
图12和13分别显示了实施例1中获得的野生型BC-PC-PLC毕赤酵母表达菌株G15和1-3，实施例6中获得的BC-PC-PLC点突变毕赤酵母表达菌株6-6和7-6以及实施例8中获得的18个BC-PC-PLC点突变毕赤酵母表达菌株的热稳定性。
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为蜡样芽胞杆菌的野生型磷脂酰胆碱特异性磷脂酶C的成熟肽的氨基酸序列。
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。
技术方案的详细描述
本申请提供了通过分子生物学中的突变筛选方法获得的磷脂酶C突变体及其用途。具体而言，所述磷脂酶C可以为磷脂酰胆碱特异性磷脂酶C(PC-PLC)。更具体而言，所述磷脂酰胆碱特异性磷脂酶C可以为蜡样芽胞杆菌的磷脂酰胆碱特异性磷脂酶C(BC-PC-PLC)。
第一方面，本申请提供了具有磷脂酰胆碱特异性磷脂酶C活性的多肽，所述多肽包含突变的SEQ ID No:2所示的氨基酸序列或其活性片段，其中所述突变包括将SEQ ID No:2所示的氨基酸序列的第63位的天冬酰胺进行突变。
在一些实施方案中，将SEQ ID No:2的氨基酸序列的第63位的天冬酰胺突变为丙氨酸(A)、半胱氨酸(C)、天冬氨酸(D)、谷氨酸(E)、苯丙氨酸(F)、甘氨酸(G)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、亮氨酸(L)、蛋氨酸(M)、谷氨酰胺(Q)、精氨酸(R)、苏氨酸(T)、缬氨酸(V)、色氨酸(W)或酪氨酸(Y)。在一些实施方案中，将SEQ ID No:2的氨基酸序列的第63位的天冬酰胺突变为丝氨酸(S)。
在一些实施方案中，所述突变还包括SEQ ID No:2氨基酸序列的第20位的精氨酸被组氨酸取代以及第83位的丙氨酸被天冬氨酸取代。
在一些实施方案中，所述多肽的氨基酸序列包含选自SEQ ID No:8、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48的氨基酸序列或由选自SEQ ID No:8、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48的氨基酸序列组成。在一些实施方案中，所述多肽的氨基酸序列由选自SEQ ID No:8、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48氨基酸序列组成，即，与SEQ ID No:2所示的氨基酸序列的区别仅在于上文所述的第63位的氨基酸突变。
在一些实施方案中，所述多肽的氨基酸序列包含SEQ ID No:12所示的氨基酸序列或由SEQ ID No:12所示的氨基酸序列组成。在一些实施方案中，所述多肽的氨基酸序列由SEQ ID No:12所示的氨基酸序列组成，即，与SEQ ID No:2所示的氨基酸序列的区别仅在于上文所述的第20位突变为组氨酸(H)、第63位突变为丝氨酸(S)和第83位突变为天冬氨酸(D)。
在一些实施方案中，所述多肽的氨基酸序列与SEQ ID No:2所示的氨基酸序列长度相同。
在一些实施方案中，所述多肽的氨基酸序列的长度大于SEQ ID No:2所示的氨基酸序列。在一些实施方案中，所述多肽还包含信号肽和/或前导肽。如“发明背景”部分所述，蜡样芽胞杆菌的野生型磷脂酰胆碱特异性磷脂酶C包含24个氨基酸的信号肽和14个氨基酸的前导肽，因此本申请的多肽也可以包含相同的或其他的信号肽和/或前导肽。在一些实施方案中，信号肽为α因子信号肽。本领域技术人员能够理解，本申请的多肽还可以包括其他功能元件，例如，但不限于，用于分离和纯化的标签元件(例如组氨酸标签)、选择元件(例如基于抗生素选择或荧光选择(例如绿色荧光蛋白，GFP))等。
在一些实施方案中，所述多肽的氨基酸序列的长度小于SEQ ID No:2所示的氨基酸序列。在相关实施方案中，所述多肽可以包含突变的SEQ ID No:2所示的氨基酸序列的活性片段，例如SEQ ID No:8、12、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48所示的氨基酸序列的活性片段。本文所用的术语“活性片段”表示野生型磷脂酰胆碱特异性磷脂酶C或本申请的磷脂酰胆碱特异性磷脂酶C突变体的一部分，该部分仍能保持磷脂酰胆碱特异性磷脂酶C的活性。蜡样芽胞杆菌的野生型磷脂酰胆碱特异性磷脂酶C的结构和功能位点在本领域中是已知的，因此本领域技术人员能够容易地制备出保留上述突变的多肽的活性片段，例如保留功能结构域的片段。有文献报道该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)，根据该PLC的晶体结构，第8，9个α螺旋为第140-153位氨基酸和第154-157 位氨基酸，第10个α螺旋为第171-186位氨基酸，所以预测的活性片段为1-170位氨基酸。
本申请还考虑到了第一方面中所述多肽的功能性变体。在一些实施方案中，所述功能性变体为保守型取代变体。“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“保守型取代变”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。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，下表中的6组中的每组都包括彼此为保守型取代的氨基酸：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)；
5)异亮氨酸(I)、亮氨酸(L)、蛋氨酸(M)、缬氨酸(V)；以及
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
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)。
在一些实施方案中，所述功能性变体与母体序列(例如SEQ ID No:8、12、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48所示的氨基酸序列)的同一性或相似性为至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或更高。
第二方面，本申请提供了编码第一方面中所述的多肽的核酸分子。本申请考虑到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。
本申请还提供了核酸分子，其包含选自SEQ ID No:7、13、15、17、19、21、23、25、27、29、31、33、37、39、41、43、45或47核酸序列。
本申请还提供了核酸分子，其包含SEQ ID No:11所示的核酸序列。
应当理解，本申请的核酸分子不仅可以包含BC-PC-PLC成熟肽突变体的编码序列，还可以包含其他核酸序列。在一些实施方案中，所述其他核酸序列为编码信号肽和/或前导肽的核酸序列。在一些实施方案中，所述其他核酸序列为编码用于分离和纯化的标签元件(例如组氨酸标签)的核酸序列、编码选择元件(例如基于抗生素选择或荧光选择(例如绿色荧光蛋白，GFP))的核酸序列。本领域技术人员能够理解，所述其他核酸序列还可以为转录和/或翻译中所需的调控序列，例如启动子、增强子等。
第三方面，本申请提供了包含第二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。
第四方面，本申请提供了包含第二方面所述的核酸分子或第三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。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。
第五方面，本申请提供了第四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。
第六方面，本申请提供了第一方面所述的多肽、或第二方面所述的核酸分子编码的多肽、或第三方面所述的载体编码的多肽、或第四方面所述的细胞表达出的多肽、或第五方面所述的磷脂酶C作为磷脂酰胆碱特异性磷脂酶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。
第七方面，本申请提供了第一方面所述的多肽、或第二方面所述的核酸分子、或第三方面所述的载体、或第四方面所述的细胞在制备脱胶酶中的用途。利用多肽、核酸分子、载体或转化的细胞制备脱胶酶的方法是本领域公知的。以发酵法为例，可以将分离得到的磷脂酶C序列的DNA序列通过表达载体转化宿主细胞(例如毕赤酵母)，进行发酵罐大规模发酵培养，通过过滤得到发酵液，再通过超滤用相应缓冲液进行置换，以脱去发酵液中浓度较高的盐离子，再向超滤液中加入通用的稳定剂(如甘油)，以及金属离子锌(以硫酸锌的形式加入)。
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.菌株和质粒
菌株：毕赤酵母GS115(Invitrogen，货号C181-00)，大肠杆菌DH5a(TAKARA，货号D9057A)。
质粒：pPIC-9k(Invitrogen，货号V17520)，pAO815质粒(Invitrogen，货号V18020)，pAO-PLC质粒和pAOmu-PLC质粒为本申请的发明人构建，具体请参见下文描述。
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。
MM-卵黄筛选培养基：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％卵黄液，2％琼脂。
卵黄液制备：取鲜鸡蛋一枚，用75％酒精擦拭消毒，用消毒的镊子敲开蛋壳使蛋清流出，用无菌水冲洗蛋黄两遍，加入装有80ml无菌水的三角瓶中，混合均匀得到20％的卵黄液。
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)，0.5％甲醇(灭菌后加入)，4×10-5％D-生物素(灭菌后加入)，0.1M磷酸二氢钾-磷酸氢二钾缓冲液(pH6.0)。
3.钼蓝法检测PLC活力所用试剂：
PLC反应液：0.5％大豆磷脂，25mM Tris-HCl pH 7.5，5mM CaCl2
CIAP反应液：50mM Tris-HCl pH 9.0，10mM MgCl2，1U CIAP(购自宝生物工程(大连)有限公司)
钼蓝显色反应液：100μl CIAP反应物，0.2％抗坏血酸，0.1％钼酸铵(用30％H2SO4配制)
4.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)
5.酶：
限制性内切酶HindIII，EcoRI，AatII(购自纽英伦生物技术(北京)有限公司)
PCR酶：TaKaRa Taq，
Figure PCTCN2014090213-appb-000001
DNA聚合酶(购自宝生物工程(大连)有限公司)
T4DNA连接酶(购自富酶泰斯有限公司)
实施例1：野生型BC-PC-PLC毕赤酵母表达菌株的构建
根据蜡样芽孢杆菌的磷脂酰胆碱特异性磷脂酶C的成熟肽序列(PDB ID:1AH7)以及毕赤酵母密码子偏好性，设计得到BC-PC-PLC的DNA序列(SEQ ID No:1)，并在其前端融合α因子信号肽序列(其DNA序列来源于商品化毕赤酵母表达载体pPIC-9k，SEQ ID No:3中第8-274位)以及毕赤酵母的Kozak序列(SEQ ID No:3中第1-7位)，最终得到α-BC-PC-PLC DNA序列(SEQ ID No:3)。
将α-BC-PC-PLC DNA序列提供给上海生工生物有限公司进行全基因合成，得到含α-BC-PC-PLC DNA序列的克隆载体pGEM-T-PLC。以此载体为模板，使用
Figure PCTCN2014090213-appb-000002
DNA聚合酶和引物对AmPLC-3/AmPLC-4，通过PCR扩增得到PLC片段。
以pPIC-9k表达载体为模板，使用
Figure PCTCN2014090213-appb-000003
DNA聚合酶和引物对AmPLC-1/AmPLC-2，通过PCR扩增得到AOX1启动子片段(PAOX1)。
通过重叠PCR，利用引物对AmPLC-1/AmPLC-4和
Figure PCTCN2014090213-appb-000004
DNA聚合酶，得到PAOX1+PLC融合片段。
利用AatII和EcoRI酶切位点将PAOX1+PLC融合片段克隆至pAO815载体中，得到表达载体pAO-PLC。将pAO-PLC用SalI线性化，凝胶回收到约8.5kb片段。利用LiAC法制备毕赤酵母GS115菌株的感受态细胞，通过电转化将线性化的pAO-PLC片段转化入GS115感受态细胞。将转化物接种于MGYS平板上，30℃培养3天。挑取平板上的单克隆，将其悬浮于5μl无菌水中。取0.5μl接种于MM-卵黄筛选平板上。30℃培养3天后，观察在菌体周围可看到白色沉淀圈的阳性克隆。表达的磷酸酶C能将卵磷脂分解为磷脂酰胆碱与非水溶性甘油二酸酯，MM-卵黄筛选平板菌落周围出现的白色沉底圈属于上述反应。菌落的磷脂酶C分泌量多或者分泌的磷脂酶C比酶活高，则菌落周围形成的白色沉淀圈相对较大。
筛选得到两株阳性菌株，如图1所示，分别指定为G15和1-3，G15的白色沉淀圈较小，1-3的白色沉淀圈较大。
实施例2：BC-PC-PLC突变体文库构建及筛选
以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000005
DNA聚合酶和引物对AmPLC-1/AOXH-2，通过PCR扩增得到约900bp片段。以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000006
DNA聚合酶和引物对AOXH-3/AmPLC-4，通过PCR扩增得到约1.1kb片段，以前两步PCR得到的约900bp片段和约1.1kb片段混合作为第三步PCR的模板，使用引物对AmPLC-1/AmPLC-4和
Figure PCTCN2014090213-appb-000007
DNA聚合酶，通过PCR扩增得到约1.9kb片段。
将该约1.9kb片段通过AatII和EcoRI酶切位点克隆至pAO-PLC中，得到pmAO-PLC。在pmAO-PLC中，pAO-PLC中的一个HindIII酶切位点被进行了突变，从而只保留位于BC-PC-PLC序列5’端的一个HindIII酶切位点，从而能使用HindIII和EcoRI将BC-PC-PLC的突变片段克隆至pmAO-PLC。
以pAO-PLC载体为模板，使用TaKaRa Taq酶和引物对EPPLC-1/EPPLC-2进行易错PCR(在PCR时额外添加0.3mM的MnCl2)，得到大小为约755bp的突变扩增子片段集合。通过HindIII和EcoRI酶切位点将得到的片段克隆至pmAO-PLC，并将得到的载体转化入大肠杆菌DH5α菌株，一共得到1×104个BC-PC-PLC突变体。
将每1×103个BC-PC-PLC突变体用2ml的无菌水洗至8ml的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(使用尽量少的DNA，保证大多数阳性转化子含单拷贝PLC基因)，用电转化法将载体转化至毕赤酵母GS115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BC-PC-PLC的毕赤酵母突变体文库。挑取平板上的单克隆，将其悬浮于5μl无菌水中，取0.5μl接种于MM-卵黄筛选平板上。以G15为对照，比较突变体和G15中白色沉淀圈的大小。最终筛选得到一株白色沉淀圈比G15大的突变体菌株，指定为7-3-3，如图2所示。
实施例3：BC-PC-PLC突变体序列分析
将7-3-3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7-3-3菌株的基因组DNA为模板，使用
Figure PCTCN2014090213-appb-000008
DNA聚合酶和引物对AOX1-5/AOX1-3进行PCR扩增，得到7-3-3菌株中BC-PC-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。7-3-3的BC-PC-PLC的DNA测序结果如SEQ ID No:4所示。经过比对发现，与SEQ ID No:3相比，SEQ ID No:4中有7个碱基发生了突变，其中包括三个有义突变，分别是第59位的G突变为A，使得第20位精氨酸突变为组氨酸(CGT→CAT)；第188位A突变为G，使得第63位天冬酰胺突变为丝氨酸(AAC→AGC)；第248位C突变为A，使得第83位丙氨酸突变为天冬氨酸(GCC→GAC)。
实施例4：BC-PC-PLC单点突变体毕赤酵母表达菌株构建及筛选
实施例4.1：PLC-R20H的构建及筛选
以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000009
DNA聚合酶和引物对EPPLC-1/20RH-2，通过PCR扩增得到约78bp片段。以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000010
DNA聚合酶和引物对20RH-3/EPPLC-2，通过PCR扩增得到约707bp片段。再以前两步PCR得到的约78bp片段和约707bp片段混合作为第三步PCR的模板，使用引物对EPPLC-1/EPPLC-2和
Figure PCTCN2014090213-appb-000011
DNA聚合酶，通过PCR扩增得到约755bp片段。
将该约755bp片段通过HindIII和EcoRI酶切位点克隆至pmAO-PLC中，得到pmAO-PLC-R20H载体。将pmAO-PLC-R20H用SalI线性化，凝胶回收8.5kb片段。利用LiAC法制备毕赤酵母GS115菌株的感受态细胞，再通过电转化将500ng线性化的pmAO-PLC-R20H转化至GS115感受态细胞。将转化物接种于MGYS平板上，30℃培养3天。挑取平板上的单克隆，将其悬浮于5μl无菌水中，取0.5μl接种于MM-卵黄筛选平板上。以G15和1-3为对照，比较突变体和G15及1-3白色沉淀圈的大小，如图3所示。在筛选平板上没有白色沉淀圈的为阴性克隆，即该克隆的基因组中未成功转入PLC基因片段。在挑选用于后续实验的突变体阳性克隆时，首选确定所有阳性克隆中比例最大且白色沉淀圈大小相同或相近的那些克隆，然后从中随机挑选一个指定为用于后续实验的PLC-R20H。这样挑选的突变体克隆往往含有单拷贝的PLC基因，便于减少表达量差异对酶活比较的影响。
实施例4.2：PLC-N63S的构建及筛选
以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000012
DNA聚合酶和引物对EPPLC-1/63NS-2，通过PCR扩增得到约207bp片段。以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000013
DNA聚合酶和引物对63NS-3/EPPLC-2，通过PCR扩增得到约576bp片段。再以前两步PCR得到的约207bp片段和约576bp片段混合作为第三步PCR的模板，使用引物对EPPLC-1/EPPLC-2和
Figure PCTCN2014090213-appb-000014
DNA聚合酶，通过PCR扩增得到约755bp片段。
将该约755bp片段通过HindIII和EcoRI酶切位点克隆至pmAO-PLC中，得到pmAO-PLC-N63S载体，将pmAO-PLC-N63S用SalI线性化，凝胶回收8.5kb片段。利用LiAC法制备毕赤酵母GS115菌株的感受态细胞，再通过电转化将500ng线性化的pmAO-PLC-N63S转化GS115感受态细胞。将转化物接种于MGYS平板上，30℃培养3天。挑取平板上的单克隆，将其悬浮于5μl无菌水中，取0.5μl接种于MM-卵黄筛选平板上。以G15和1-3为对照，比较突变体和G15及1-3白色沉淀圈的大小，如图4所示。如实施例4.1中所述，选取用于后续实验的突变体阳性克隆PLC-N63S。
实施例4.3：PLC-A83D的构建及筛选
以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000015
DNA聚合酶和引物对EPPLC-1/83AD-2，通过PCR扩增得到约266bp片段。以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000016
DNA聚合酶和引物对83AD-3/EPPLC-2，通过PCR扩增得到约520bp片段。再以前两步PCR得到的约266bp片段和约502bp片段混合作为第三步PCR的模板，使用引物对EPPLC-1/EPPLC-2和
Figure PCTCN2014090213-appb-000017
DNA聚合酶，通过PCR扩增得到约755bp片段。
将该约755bp片段通过HindIII和EcoRI酶切位点克隆至pmAO-PLC中，得到pmAO-PLC-A83S载体。将pmAO-PLC-A83S用SalI线性化，凝胶回收8.5kb片段。利用LiAC法制备毕赤酵母GS115菌株的感受态细胞，再通过电转化将500ng线性化的pmAO-PLC-A83D转化至GS115感受态细胞。将转化物接种于MGYS平板上，30℃培养3天。挑取平板上的单克隆，将其悬浮于5μl无菌水中，取0.5μl接种于MM-卵黄筛选平板上。以G15和1-3为对照，比较突变体和G15及1-3白色沉淀圈的大小，如图5所示。如实施例4.1中所述，选取用于后续实验的突变体阳性克隆PLC-A83D。
实施例4.4：PLC-R20HN63SA83D的构建及筛选
以7-3-3菌株的基因组DNA为模板，使用
Figure PCTCN2014090213-appb-000018
DNA聚合酶和引物对EPPLC-1/EPPLC-2，通过PCR扩增得到约755bp片段。
将该片段通过HindIII和EcoRI克隆至pmAO-PLC中，得到pmAO-PLC-R20HN63SA83D载体。将pmAO-PLC-R20HN63SA83D用SalI线性化，凝胶回收8.5kb片段。利用LiAC法制备毕赤酵母GS115菌株的感受态细胞，再通过电转化将500ng线性化的pmAO-PLC-R20HN63SA83D转化至GS115感受态细胞。将转化物接种于MGYS平板上，30℃培养3天。挑取平板上的单克隆，将其悬浮于5μl无菌水中，取0.5μl接种于MM-卵黄筛选平板上。以G15和1-3为对照，比较突变体和G15及1-3白色沉淀圈的大小，如图6所示。如实施例4.1中所述，选取用于后续实验的突变体阳性克隆PLC-R20HN63SA83D。
PLC-N63S突变体在筛选平板上的白色沉淀圈基本与PLC-R20HN63SA83D克隆的白色沉淀圈相当，并且明显比G15和1-3的白色沉淀圈更大。PLC-R20H和PLC-A83D突变体在白色沉淀圈上的大小基本与G15和1-3的白色沉淀圈大小相当。从而证实：第63位氨基酸从天冬酰胺突变为丝氨酸能够使BC-PC-PLC的酶活力提高。
实施例5：BC-PC-PLC突变体菌株的摇瓶发酵
取G15、1-3、PLC-R20H、PLC-N63S、PLC-A83D和PLC-R20HN63SA83D菌株，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。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)，30％甘油，0.3％ZnSO4·7H2O)。
取10μl发酵液至190μl PLC反应液(含0.5％大豆磷脂，25mM Tris-HCl pH 7.5，5mM CaCl2)中，37℃振荡孵育30min。孵育后，加入100μl氯仿振荡混匀，12000rpm离心2min。取80μl上清，加入20μl CIAP反应液(含50mM Tris-HCl pH 9.0，10mM MgCl2，1U CIAP)，37℃振荡孵育1h。
孵育后，取25μl反应物，加入975μl钼蓝显色液(含0.2％抗坏血酸，0.1％钼酸铵)，37℃振荡孵育10min。在700nm波长下测定样品的吸光度，计算得到各个发酵液样品的PLC活力。用Bradford试剂测定G15、1-3、PLC-R20H、PLC-N63S、PLC-A83D和PLC-R20HN63SA83D菌株的摇瓶发酵液的蛋白浓度，从而得出：G15、1-3、PLC-R20H、PLC-N63S、PLC-A83D和PLC-R20HN63SA83D的比酶活。如图7所示，PLC-N63S和PLC-R20HN63SA83D的比酶活是野生型的约4倍，PLC-R20H和PLC-A83D的比酶活与野生型相当，证明第63位天冬酰胺突变为丝氨酸是比酶活提高的关键突变位点。
将G15、1-3、PLC-R20H、PLC-N63S、PLC-A83D和PLC-R20HN63SA83D菌株的摇瓶发酵液的蛋白量调至相同量进行SDS-PAGE电泳，结果如图8所示。PLC-N63S PLC蛋白条带强度略少于G15的PLC蛋白条带强，因此PLC-N63S的比酶活至少是G15的4倍。
实施例6：PLC-N63S和PLC-R20HN63SA83D高产菌株构建及摇瓶发酵
将pmAO-PLC-N63S和pmAO-PLC-R20HN63SA83D用SalI线性化，凝胶回收约8.5kb片段。利用LiAC法制备毕赤酵母GS115菌株的感受态细胞，再通过电转化将2μg线性化的pmAO-PLC-R20HN63SA83D转化至GS115感受态细胞。将转化物接种于MGYS平板上，30℃培养3天。挑取平板上的单克隆，将其悬浮于5μl无菌水中，取0.5μl接种于MM-卵黄筛选平板上。从100个PLC-N63S转化子中挑出11个白色沉淀圈最大的转化子，命名为6-1至6-11。同样，从100个PLC-R20HN63SA83D转化子中挑出6个白色沉淀圈最大的转化子，命名为7-1至7-6。
取6-1至6-11和7-1至7-6菌株，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。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)，30％甘油，0.3％ ZnSO4·7H2O)。
用Bradford试剂测定6-1至6-11和7-1至7-6菌株的摇瓶发酵液的蛋白浓度，结果如图9所示。
在6-1至6-11菌株中发酵液蛋白浓度最高的是6-6菌株，发酵液蛋白浓度为0.108mg/ml，而7-1至7-6中发酵液蛋白浓度最高的是7-6菌株，发酵液蛋白浓度为0.238mg/ml。取两种菌株中蛋白浓度较高的几株菌进行SDS-PAGE电泳，比较PLC目的条带蛋白量，结果如图10所示，7-5和7-6的PLC目的条带强度高于6-2、6-3、6-6、6-9、6-11的PLC目的条带量。从而说明PLC-R20HN63SA83D突变体中的R20H和A83D突变有助于提高突变体在毕赤酵母中的表达量。
实施例7：BC-PC-PLC第63位氨基酸饱和突变
将BC-PC-PLC第63位天冬酰胺分别突变为丙氨酸(A)，半胱氨酸(C)，天冬氨酸(D)，谷氨酸(E)，苯丙氨酸(F)，甘氨酸(G)，组氨酸(H)，异亮氨酸(I)，赖氨酸(K)，亮氨酸(L)，甲硫氨酸(M)，脯氨酸(P)，谷氨酰胺(Q)，精氨酸(R)，苏氨酸(T)，结氨酸(V)，色氨酸(W)和酪氨酸(Y)。
简单而言，以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000019
DNA聚合酶和引物对EPPLC-1/63X-2(X代表上述18种氨基酸的单字母简写)，通过PCR扩增得到约207bp片段。以pAO-PLC载体为模板，使用
Figure PCTCN2014090213-appb-000020
DNA聚合酶和引物对63X-3/EPPLC-2，通过PCR扩增得到约576bp片段。再以前两步PCR得到的约207bp片段和约576bp片段混合作为第三步PCR的模板，使用引物对EPPLC-1/EPPLC-2和
Figure PCTCN2014090213-appb-000021
DNA聚合酶，通过PCR扩增得到约755bp片段。
将得到的18个约755bp片段分别通过HindIII和EcoRI酶切位点克隆至pmAO-PLC中，得到18种pmAO-PLC-N63X载体，将18种pmAO-PLC-N63X载体用SalI线性化，凝胶回收8.5kb片段。利用LiAC法制备毕赤酵母GS115菌株的感受态细胞，再通过电转化将500ng线性化的18种pmAO-PLC-N63X分别转化GS115感受态细胞。将转化物接种于MGYS平板上，30℃培养3天。挑取平板上的单克隆，将其悬浮于5μl无菌水中，取0.5μl接种于MM-卵黄筛选平板上。以G15和1-3为对照，比较突变体和G15及1-3白色沉淀圈的大小。如实施例4.1中所述，选取用于后续实验的突变体阳性克隆PLC-N63X。其中PLC-N63P，即第63位氨基酸突变为脯氨酸的突变体无白色沉淀圈形成，表明该突变使PLC失活。
实施例8：BC-PC-PLC饱和突变体菌株的摇瓶发酵酶活及热稳定性
取G15、1-3、6-6、7-6、PLC-N63A、PLC-N63C、PLC-N63D、PLC-N63E、PLC-N63F、PLC-N63G、PLC-N63H、PLC-N63I、PLC-N63K、PLC-N63L、PLC-N63M、PLC-N63P、 PLC-N63Q、PLC-N63R、PLC-N63T、PLC-N63V、PLC-N63W、PLC-N63Y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。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)，30％甘油，0.3％ZnSO4·7H2O)。
取10μl发酵液至190μl PLC反应液(含0.5％大豆磷脂，25mM Tris-HCl pH 7.5，5mM CaCl2)中，37℃或者60℃振荡孵育30min。孵育后，加入100μl氯仿振荡混匀，12000rpm离心2min。取80μl上清，加入20μl CIAP反应液(含50mM Tris-HCl pH 9.0，10mM MgCl2，1U CIAP)，37℃振荡孵育1h。
孵育后，取25μl反应物，加入975μl钼蓝显色液(含0.2％抗坏血酸，0.1％钼酸铵)，37℃振荡孵育10min。在700nm波长下测定样品的吸光度，计算得到各个发酵液样品的PLC活力。
用Bradford试剂测定G15、1-3、6-6(PLC-N63S)、7-6、PLC-N63A、PLC-N63C、PLC-N63D、PLC-N63E、PLC-N63F、PLC-N63G、PLC-N63H、PLC-N63I、PLC-N63K、PLC-N63L、PLC-N63M、PLC-N63P、PLC-N63Q、PLC-N63R、PLC-N63T、PLC-N63V、PLC-N63W、PLC-N63Y发酵液蛋白浓度。
从而得到各个突变体的在37℃和60℃反应的比酶活，如图11所示，从图中可知，在37℃反应时，PLC-N63P几乎没有活性，PLC-N63C、PLC-N63D、PLC-N63E和PLC-N63I比酶活提高效果不明显外，其余突变体的比酶活均比野生型提高1倍以上，其中PLC-N63S、PLC-N63A、PLC-N63F、PLC-N63H、PLC-N63K、PLC-N63R、PLC-N63T、PLC-N63W和PLC-N63Y的比酶活是野生型的4倍以上。
在60℃反应时，PLC-N63P几乎没有活性，PLC-N63D和PLC-N63I比酶活提高效果不明显外，其余突变体的比酶活均比野生型提高1倍以上，其中PLC-N63S、PLC-N63A、PLC-N63F、PLC-N63H、PLC-N63K、PLC-N63R、PLC-N63W和PLC-N63Y的比酶活是野生型的6倍以上。
野生型在60℃反应时的比酶活是在37℃反应时的1.5倍，而突变体PLC-N63S、PLC-N63A、PLC-N63C、PLC-N63D、PLC-N63E、PLC-N63F、PLC-N63G、PLC-N63H、PLC-N63I、PLC-N63K、PLC-N63L、PLC-N63M、PLC-N63R、PLC-N63V、PLC-N63W和PLC-N63Y在60℃反应时的比酶活是在37℃反应时的2倍以上。说明突变体PLC-N63S、PLC-N63A、PLC-N63C、PLC-N63D、PLC-N63E、PLC-N63F、PLC-N63G、PLC-N63H、PLC-N63I、PLC-N63K、PLC-N63L、PLC-N63M、PLC-N63R、PLC-N63V、PLC-N63W和PLC-N63Y比野生型更适合在60℃进行反应。
将所有发酵液在90℃的条件下孵育1h后，分别在37℃和60℃条件下进行反应测定经过90℃处理后的比酶活。如图12所示，突变体PLC-N63F、PLC-N63W和PLC-N63Y经过在90℃的条件下处理1h后在37℃反应均仍然保留50％的活力，而野生型和其余突变体的比酶活大大降低。如图13所示，突变体PLC-N63F、PLC-N63W和PLC-N63Y经过在90℃的条件下处理1h后在60℃反应均仍然保持有30％的活力，而野生型和其余突变体的比酶活大大降低。说明PLC-N63F、PLC-N63W和PLC-N63Y具有良好的热稳定性。
由以上实施例可见，BC-PC-PLC的第63位氨基酸由天冬酰胺突变为丙氨酸(A)，苯丙氨酸(F)，甘氨酸(G)，组氨酸(H)，异亮氨酸(I)，赖氨酸(K)，亮氨酸(L)，甲硫氨酸(M)，谷氨酰胺(Q)，精氨酸(R)，苏氨酸(T)，丝氨酸(S)，结氨酸(V)，色氨酸(W)和酪氨酸(Y)后，37℃和60℃的比酶活比野生型有2倍以上的提高。突变为苯丙氨酸(F)，色氨酸(W)和酪氨酸(Y)后突变体变现出良好的热稳定性，经过在90℃的条件下处理1h后，37℃反应时的比酶活均能保留约50％，60℃反应时的比酶活均能保留约30％，优于野生型和其他突变体。此外BC-PC-PLC的第20位精氨酸突变为组氨酸以及第83位的丙氨酸突变为天冬氨酸，能够使得第63位氨基酸由天冬酰胺突变为丝氨酸时的突变体在毕赤酵母中的表达量提高。
序列描述
SEQ ID No:1：野生型BC-PC-PLC DNA编码序列；
SEQ ID No:2：野生型BC-PC-PLC氨基酸序列；
SEQ ID No:3：人工合成的a-BC-PC-PLC DNA序列；
SEQ ID No:4：BC-PC-PLC突变体7-3-3DNA编码序列；
SEQ ID No:5：突变体PLC-R20H DNA编码序列；
SEQ ID No:6：突变体PLC-R20H氨基酸序列；
SEQ ID No:7：突变体PLC-N63S DNA编码序列；
SEQ ID No:8：突变体PLC-N63S氨基酸序列；
SEQ ID No:9：突变体PLC-A83D DNA编码序列；
SEQ ID No:10：突变体PLC-A83D氨基酸序列；
SEQ ID No:11：PLC-R20HN63SA83D DNA编码序列；
SEQ ID No:12：PLC-R20HN63SA83D氨基酸序列；
SEQ ID No:13：PLC-N63A DNA编码序列；
SEQ ID No:14：PLC-N63A氨基酸序列；
SEQ ID No:15：PLC-N63C DNA编码序列；
SEQ ID No:16：PLC-N63C氨基酸序列；
SEQ ID No:17：PLC-N63D DNA编码序列；
SEQ ID No:18：PLC-N63D氨基酸序列；
SEQ ID No:19：PLC-N63E DNA编码序列；
SEQ ID No:20：PLC-N63E氨基酸序列；
SEQ ID No:21：PLC-N63F DNA编码序列；
SEQ ID No:22：PLC-N63F氨基酸序列；
SEQ ID No:23：PLC-N63G DNA编码序列；
SEQ ID No:24：PLC-N63G氨基酸序列；
SEQ ID No:25：PLC-N63H DNA编码序列；
SEQ ID No:26：PLC-N63H氨基酸序列；
SEQ ID No:27：PLC-N63I DNA编码序列；
SEQ ID No:28：PLC-N63I氨基酸序列；
SEQ ID No:29：PLC-N63K DNA编码序列；
SEQ ID No:30：PLC-N63K氨基酸序列；
SEQ ID No:31：PLC-N63L DNA编码序列；
SEQ ID No:32：PLC-N63L氨基酸序列；
SEQ ID No:33：PLC-N63M DNA编码序列；
SEQ ID No:34：PLC-N63M氨基酸序列；
SEQ ID No:35：PLC-N63P DNA编码序列；
SEQ ID No:36：PLC-N63P氨基酸序列；
SEQ ID No:37：PLC-N63Q DNA编码序列；
SEQ ID No:38：PLC-N63Q氨基酸序列；
SEQ ID No:39：PLC-N63R DNA编码序列；
SEQ ID No:40：PLC-N63R氨基酸序列；
SEQ ID No:41：PLC-N63T DNA编码序列；
SEQ ID No:42：PLC-N63T氨基酸序列；
SEQ ID No:43：PLC-N63V DNA编码序列；
SEQ ID No:44：PLC-N63V氨基酸序列；
SEQ ID No:45：PLC-N63W DNA编码序列；
SEQ ID No:46：PLC-N63W氨基酸序列；
SEQ ID No:47：PLC-N63Y DNA编码序列；
SEQ ID No:48：PLC-N63Y氨基酸序列。
引物序列列表
Figure PCTCN2014090213-appb-000022
Figure PCTCN2014090213-appb-000023

Claims (10)

  1. 具有磷脂酰胆碱特异性磷脂酶C活性的多肽，所述多肽包含突变的SEQ ID No:2所示的氨基酸序列或其活性片段，其中所述突变包括将SEQ ID No:2所示的氨基酸序列的第63位的天冬酰胺进行突变。
  2. 如权利要求1所述的多肽，其中将SEQ ID No:2的氨基酸序列的第63位的天冬酰胺突变为丝氨酸(S)、丙氨酸(A)、苯丙氨酸(F)、组氨酸(H)、赖氨酸(K)、精氨酸(R)、色氨酸(W)、酪氨酸(Y)、半胱氨酸(C)、天冬氨酸(D)、谷氨酸(E)、甘氨酸(G)、异亮氨酸(I)、亮氨酸(L)、蛋氨酸(M)、谷氨酰胺(Q)、苏氨酸(T)或缬氨酸(V)。
  3. 如权利要求1或2所述的多肽，其中所述突变还包括SEQ ID No:2氨基酸序列的第20位的精氨酸被组氨酸取代以及第83位的丙氨酸被天冬氨酸取代，优选地，所述多肽的氨基酸序列包含SEQ ID No:12所示或由SEQ ID No:12所示的氨基酸序列组成。
  4. 如权利要求1或2所述的多肽，所述多肽的氨基酸序列包含选自SEQ ID No:8、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48的氨基酸序列或由选自SEQ ID No:8、14、16、18、20、22、24、26、28、30、32、34、38、40、42、44、46或48的氨基酸序列组成。
  5. 编码权利要求1-4中任一项所述的多肽的核酸分子，优选地，所述核酸分子包含选自SEQ ID No:7、11、13、15、17、19、21、23、25、27、29、31、33、37、39、41、43、45或47的核酸序列。
  6. 包含权利要求5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，进一步优选地，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。
  7. 包含权利要求5所述的核酸分子或权利要求6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。
  8. 使用权利要求7所述的细胞产生的磷脂酶C。
  9. 权利要求1-4中任一项所述的多肽、或权利要求5所述的核酸分子编码的多肽、或权利要求6所述的载体编码的多肽、或权利要求7所述的细胞表达出的多肽、或权利要求8所述的磷脂酶C作为磷脂酰胆碱特异性磷脂酶C的用途，优选为在油脂脱胶工艺中的用途。
  10. 权利要求1-4中任一项所述的多肽、或权利要求5所述的核酸分子、或权利要求6所述的载体、或权利要求7所述的细胞在制备脱胶酶中的用途。
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