CN108384768A - A kind of mutant of phospholipase C and its application - Google Patents

A kind of mutant of phospholipase C and its application Download PDF

Info

Publication number
CN108384768A
CN108384768A CN201810166718.6A CN201810166718A CN108384768A CN 108384768 A CN108384768 A CN 108384768A CN 201810166718 A CN201810166718 A CN 201810166718A CN 108384768 A CN108384768 A CN 108384768A
Authority
CN
China
Prior art keywords
sequence
phospholipase
oil
mutant
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810166718.6A
Other languages
Chinese (zh)
Inventor
严明
陈圣
章志林
魏淼
陈晶晶
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing New Enzyme Biological Technology Co Ltd
Jiangsu Zhongmei Biological Technology Co Ltd
Original Assignee
Nanjing New Enzyme Biological Technology Co Ltd
Jiangsu Zhongmei Biological Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing New Enzyme Biological Technology Co Ltd, Jiangsu Zhongmei Biological Technology Co Ltd filed Critical Nanjing New Enzyme Biological Technology Co Ltd
Priority to CN201810166718.6A priority Critical patent/CN108384768A/en
Publication of CN108384768A publication Critical patent/CN108384768A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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)

Abstract

The present invention relates to mutant and its application of a kind of phospholipase C.The mutant for the phospholipase C with PC, PE and PI activity specific that the present invention is improved using rite-directed mutagenesis, can be effectively used for enzymatic degumming.The present invention also includes constructs, carrier and the host cell of the polynucleotides and the polynucleotides that encode the mutant enzyme, together with the method for producing the mutant and using the mutant.

Description

A kind of mutant of phospholipase C and its application
The reference of sequence table
The application includes the sequence table of computer-reader form, is incorporated herein by reference.
Technical field
The invention belongs to biotechnology, it is related to mutant and its application of a kind of phospholipase C, specifically, being to relate to And a kind of phospholipase C with PC, PE and PI activity specific mutant and its be applied to enzymatic degumming.
Background technology
Degumming is an important step of oil and fat refining, and traditional aquation method for degumming economic cost is high, and material breakage is big, Environmental pollution is serious.In recent years, many people were dedicated to enzymatic degumming for the degumming link in oil and fat refining, and achieved very Big progress.Enzymatic degumming has mild condition, the advantages such as pollution-free, oil quality is high compared to conventional method.Used in fat degumming Enzyme be phosphatidase, it is known that a few types phosphatidase, according to the position of its key attacked in phospholipid molecule in Qi Te Anisotropic aspect is different.Phospholipase A1 (PLA1), phospholipase A2 (PLA2) can special hydrolytic phosphatide glycerine Sn-1 or Ester bond on the positions Sn-2 generates lysophosphatide and free fatty, to remove non-hydratable phospholipid.Term phospholipase B (PLB) The phosphatidase that be used to have both A1 activity and A2 activity.Phospholipase C (PLC) mainly acts on glycerine on the positions glycerophosphatide C3 Phosphoric acid ester bond generates 1,2- diacylglycerols and phosphate (phosphocholine, phosphoethanolamine, phosphoserine and phosphoinositide Deng).Phospholipase D (PLD) generates 1,2- diacylglycerols-phosphate and base groups, as shown in Figure 1.
About the summary to enzymatic degumming, referring to Di Jiesitela (Dijkstra) 2010, European lipid Science and Technology Magazine (Eur.] .Lipid Sci.Technol.) 112,1178.The purposes of phospholipase A and/or phospholipase C in degumming is for example Be described in a gram Lawson (Clausen) 2001, European lipid Science and Technology magazine 103 333-340, WO2003/089620 and In WO2008/094847.Phospholipase A solution generates lysophosphatide and free fatty, leads to oily loss.Come from another point of view It says, phospholipase C has following advantage:It produce be all lubricant component diglyceride, as shown in Fig. 2, therefore by reduce damage It loses.There are four kinds of main phospholipids, phosphatidyl choline (PC), phosphatidyl-ethanolamine (PE), phosphatidic acid (PA) and phosphatide in vegetable oil Acyl inositol (PI).Phospholipase C enzyme has different specificity to these phosphatide.Unique known commercially available phospholipase C is model The Purifine (Di Jiesitela, the 101st AOCS meeting, on May 10th, 2010) of grace Nimes/DSM N. V., to PC and PE With specificity.WO 07/059927 describes the heat-resisting bud for degumming from Bacillus PLC.WO2012/062817 is described There is the fungi PLC of specificity to all four phosphatide.PI specific phospholipases C has been described in WO 2011/046815. CN 106103704A and CN 106459936A describe the phospholipase C with PI specificity and PC and PE specificity.
PLC expression quantity in animal and plant body is extremely limited, and extraction and separation process are cumbersome, of high cost.It is microbe-derived The PLC in the genetic engineering bacterium source especially after recombination has many advantages, such as with short production cycle, and yield is big.But still also face one A little unavoidable problems, such as：It is pathogenic bacteria mostly to produce PLC original bacterias, and there are security risks for the application in terms of food；It compares In traditional acid system degumming, enzymatic degumming still suffers from the higher problem of cost；In addition, the PLC in common micro-organisms source is to acid It is undesirable with the tolerance of heat, its application in food industry is greatly limited, therefore needs to develop or be transformed to have provided higher The PLC of active and stronger acidproof and heat resistance improves the enzyme hydrolysis efficiency of phosphatide, to improve production economy benefit.
Description of the drawings
Fig. 1 is that different phosphatidases cut the place of phosphatide together with four kinds of main functional groups on phosphatide；
Fig. 2 is that phosphatide and phospholipase C are reacted to form diglyceride and phosphate or phosphoric acid.
Invention content
The present invention is transformed the phospholipase C with PC, PE and PI- activity specific using rite-directed mutagenesis, improves it The efficiency of hydrolytic phosphatide in enzymatic degumming.
In order to achieve the above objects and other related objects, the present invention provides a kind of phospholipase C mutant, the phospholipase C Mutant be the phospholipase C mutant with PC, PE and PI- activity specific, contain selected from amino acid as follows One of sequence：
(a)、SEQ ID No：1 to SEQ ID No：Sequence shown in 5；
(b), with sequence shown in (a) at least 90% homogeneity and with the sequence of improved phospholipase C activity；
(c), the sequence in (a) is obtained having and be improved through missing, insertion and/or the one or more amino acid residues of substitution Phospholipase C activity sequence；
Wherein sequence shown in (b) is not SEQ ID No：Sequence shown in 6.
In one embodiment, the phospholipase C mutant, containing such as SEQ ID No：1, shown in 2,3,4 or 5 Amino acid sequence.
In one embodiment, the phospholipase C mutant, containing such as SEQ ID No：Amino acid sequence shown in 2 Row.
The invention further relates to the nucleotide coding sequences of phospholipase C mutant as described above, containing as follows Sequence：
(a)、SEQ ID No：Sequence shown in 8-12；
(b), it at least 90% homogeneity and is encoded with improved phospholipase C activity with the sequence described in (a) The sequence of polypeptide or protein；
(c), hybridize under high stringency conditions with the sequence described in (a) and encode with improved phospholipase C activity The sequence of polypeptide or protein；
Wherein, the nucleotide coding sequence does not contain SEQ ID No：Sequence shown in 7.
In one embodiment, the nucleotide coding sequence contains such as SEQ ID No：Sequence shown in 9.
The present invention is also related to include nucleic acid construct, expression vector and the recombinant host cell of the polynucleotides：And it relates to And the method for generating polynucleotides.
The present invention provides the method for reducing content of phospholipid in fluid composition, this method includes
(a) a kind of fluid composition for including a certain amount of phosphatide is provided,
(b) under conditions of being enough that the enzyme is made to react with the phosphatide, the fluid composition and a kind of PC, PE and PI is special Property the contact of active phospholipase C mutant, to generate diglyceride and phosphate；And
(c) phosphate is detached from the fluid composition.
The present invention is with from the phosphatidase with PC, PE and PI- activity specific of Labilithrix luteola bacterium C genes (such as SEQ ID No：Shown in 7) it is the gene that sets out, gene mutation is carried out, activity raising is obtained by directed screening Phospholipase C mutant.
The phosphatidase with PC, PE and PI- activity specific from Labilithrix luteola bacterium of the present invention The amino acid sequence of the mutant of C contains following sequence：
(1)SEQ ID No：Amino acid sequence shown in 1：Mutational site is D42E：
MALHRSLLALLVASLASLIACAAPSDGSDDGEEVDNQEAHWEAQSVTNESESTHLWIVDRGIDILAHHG DSDPVAARAWGLMTNATCRAQWQQGLYDADFKAAYNNGRSDLPPNPSDVQVGLAGATWASHFYDPDTGKNYKGETSP TAYSEASAHLSLAKENHLSDGKAKGCYELGLALHYFTDLTQPMHAANFTAVNRPAKLHSNLEGYSMELQERYPLEDW SGPPSGTTRDFLVKTAKDSKPLFMEGVEAIVAAYKSYTGWRILQCRNIDAAAWRFVERQHVDYRDCWEGNAGVDAVI GKTLRFAQERTAQYIYLVAKEIGGDAPTTSGSP
(2)SEQ ID No：Amino acid sequence shown in 2：Mutational site is D42E+L179A：
MALHRSLLALLVASLASLIACAAPSDGSDDGEEVDNQEAHWEAQSVTNESESTHLWIVDRGIDILAHHG DSDPVAARAWGLMTNATCRAQWQQGLYDADFKAAYNNGRSDLPPNPSDVQVGLAGATWASHFYDPDTGKNYKGETSP TAYSEASAHLSLAKENHLSDGKAKGCYELGLAAHYFTDLTQPMHAANFTAVNRPAKLHSNLEGYSMELQERYPLEDW SGPPSGTTRDFLVKTAKDSKPLFMEGVEAIVAAYKSYTGWRILQCRNIDAAAWRFVERQHVDYRDCWEGNAGVDAVI GKTLRFAQERTAQYIYLVAKEIGGDAPTTSGSP
(3)SEQ ID No：Amino acid sequence shown in 3：Mutational site is D42E+Q119V：
MALHRSLLALLVASLASLIACAAPSDGSDDGEEVDNQEAHWEAQSVTNESESTHLWIVDRGIDILAHHG DSDPVAARAWGLMTNATCRAQWQQGLYDADFKAAYNNGRSDLPPNPSDVVVGLAGATWASHFYDPDTGKNYKGETSP TAYSEASAHLSLAKENHLSDGKAKGCYELGLALHYFTDLTQPMHAANFTAVNRPAKLHSNLEGYSMELQERYPLEDW SGPPSGTTRDFLVKTAKDSKPLFMEGVEAIVAAYKSYTGWRILQCRNIDAAAWRFVERQHVDYRDCWEGNAGVDAVI GKTLRFAQERTAQYIYLVAKEIGGDAPTTSGSP
(4)SEQ ID No：Amino acid sequence shown in 4：Mutational site is Q119V+M212W:
MALHRSLLALLVASLASLIACAAPSDGSDDGEEVDNQEAHWDAQSVTNESESTHLWIVDRGIDILAHHG DSDPVAARAWGLMTNATCRAQWQQGLYDADFKAAYNNGRSDLPPNPSDVVVGLAGATWASHFYDPDTGKNYKGETSP TAYSEASAHLSLAKENHLSDGKAKGCYELGLALHYFTDLTQPMHAANFTAVNRPAKLHSNLEGYSWELQERYPLEDW SGPPSGTTRDFLVKTAKDSKPLFMEGVEAIVAAYKSYTGWRILQCRNIDAAAWRFVERQHVDYRDCWEGNAGVDAVI GKTLRFAQERTAQYIYLVAKEIGGDAPTTSGSP
(5)SEQ ID No：Amino acid sequence shown in 5：Mutational site is M212W+L179A:
MALHRSLLALLVASLASLIACAAPSDGSDDGEEVDNQEAHWDAQSVTNESESTHLWIVDRGIDILAHHG DSDPVAARAWGLMTNATCRAQWQQGLYDADFKAAYNNGRSDLPPNPSDVQVGLAGATWASHFYDPDTGKNYKGETSP TAYSEASAHLSLAKENHLSDGKAKGCYELGLAAHYFTDLTQPMHAANFTAVNRPAKLHSNLEGYSWELQERYPLEDW SGPPSGTTRDFLVKTAKDSKPLFMEGVEAIVAAYKSYTGWRILQCRNIDAAAWRFVERQHVDYRDCWEGNAGVDAVI GKTLRFAQERTAQYIYLVAKEIGGDAPTTSGSP
The DNA sequences encoding of above-mentioned phospholipase C mutant includes following DNA sequence dna:
(1)SEQ ID No：8, it is SEQ ID No：The GCC of 124-126bp in phospholipase C gene sequence shown in 7 Sport GAA；
(2)SEQ ID No：9, it is SEQ ID No：The GCC of 124-126bp in phospholipase C gene sequence shown in 7 GAA is sported, and the CTG of 535-537bp sports GCG；
(3)SEQ ID No：10, it is SEQ ID No：124-126bp in phospholipase C gene sequence shown in 7 GCC sports GAA, and the CAG of 355-357bp sports GTG；
(4)SEQ ID No：11, it is SEQ ID No：355-357bp in phospholipase C gene sequence shown in 7 CAG sports GTG, and the ATG of 634-636bp sports TGG；
(5)SEQ ID No：12, it is SEQ ID No：634-636bp in phospholipase C gene sequence shown in 7 ATG sports TGG, and the CTG of 535-537bp sports GCG.
Related content in said program is explained as follows：
Phospholipase C activity：" phospholipase C activity ", " PLC activity " are related to removing phosphonate moiety from phosphatide to generate 1,2- The enzymatic activity (as shown in Figure 2) of diacylglycerol.Most of PLC enzymes belong to hydrolase and di-phosphate ester enzyme family, and generally quilt It is classified as EC 3.1.4.3.Some PLC enzymes are classified into other EC classes, for example, mono- specificity PLC of PI.It can be according to following Program described in embodiment 4 determines phosphatidase by one of measurement described in " activity of phospholipase measurement " part C activity.
Phospholipase C specificity：It is related to the polypeptide with phospholipase C activity, wherein once four kinds are most importantly phosphatidyl Choline (PC), phosphatidyl-ethanolamine (PE), phosphatidic acid (PA) and phosphatidylinositols (PI), the activity are to one or more phosphatide (as shown in Figure 1) with specificity.Phospholipase C specificity can be by as described in following embodiments 432P-NMR comes It determines.
PC, PE and PI specific phospholipase C：It is related to phosphatidyl choline (PC), phosphatidyl-ethanolamine (PE) and phosphatidyl The active polypeptide of inositol (PI).
Sequence identity：Correlation between two amino acid sequences of description or between two nucleotide sequences.
For the present invention, the degree of sequence identity between two amino acid sequences is by using such as EMBOSS software packages (EMBOSS：The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends in Genetics 16：276-277) the Needleman- executed in the Needle programs of (preferably 3.0.0 editions or higher version wood) Wunsch algorithms (Needleman and Wunsch, 1970, J.Mol.Biol.48：443-453) measure.The optional parameters used It is 10 for gap open penalty (gap open penalty), gap extension penalty (gap extension penalty) is 0.5 With EBLOSUM62 (the EMBOSS versions of BLOSUM62) substitution matrix.It is labeled as " highest identity (longest using Needle Identity output result (- nobrief options is used to obtain)) " is used as homogeneity percentage, and calculates as follows：
(same residue × 100)/(sum for comparing notch during length one compares)
For the present invention, the degree of sequence identity between two nucleotide sequences is by using such as EMBOSS software packages (EMBOSS：The European Molecular Bi010Open Software Suite, Rice etc., see above) (preferably 3.0.0 version or more highest version) Needle programs in execute Needleman-Wunsch algorithms (Needleman and Wunsch, 1970, see above) it measures.The optional parameters used is that gap open penalty is 10, and gap extension penalty is 0.5 He EDNAFULL (the EMBOSS versions of NCBI NUC4.4) substitution matrix.The output knot of " highest identity " is labeled as using Needle Fruit (- nobrief options is used to obtain) is used as homogeneity percentage, and calculates as follows：
(same deoxyribonucleotide × 100)/(sum for comparing notch during length one compares)
High stringency conditions：Mean for length is the probe of at least 100 nucleotide, it then follows standard DNA trace journey Sequence, at 42 DEG C in the toadfish sperm DNA and 50% formamide that 5X SSPE, 0.3%SDS, 200 micrograms/ml are sheared and are denaturalized Prehybridization 12 to 24 hours.Carrier material finally uses 2X SSC, 0.2%SDS, is washed at 65 DEG C three times, 15 minutes every time.
Coded sequence：Mean the polynucleotides of directly specified polypeptid acid sequence.The boundary of coded sequence is usually by opening Put reading frame decision, the open reading frame usually with ATG initiation codon or alternative initiation codon such as GTG and TTG starts, and is terminated with terminator codon such as TAA, TAG and TGA.Coded sequence can be DNA, cDNA, synthesis or again The polynucleotides of group.
cDNA：Meaning can be by reverse transcription from the mRNA molecules preparation of the maturation derived from eukaryocyte, own montage DNA molecular.CDNA lacks the intron sequences being typically found in corresponding gene group DNA.It is (initial) of starting, primary RNA transcript is the precursor of mRNA, is processed by a series of step (including montage) and is then used as ripe own montage MRNA occurs.
Nucleic acid construct：Mean single-stranded or double-stranded nucleic acid molecules, is isolated from naturally occurring gene, or by it with this Mode to be not present in (not otherwise exist) nature modify with containing the section of nucleic acid or its for synthesis 's.When the nucleic acid construct contain expression the present invention coded sequence needed for regulating and controlling sequence when, term nucleic acid construct with Term " expression cassette " is synonymous.
Regulating and controlling sequence (control sequence)：Mean that the polynucleotides expression for encoding polypeptide of the present invention be required All the components.Each regulating and controlling sequence can be natural or external source for the nucleotide sequence of coding said polypeptide or each Regulating and controlling sequence is for that can be natural or external source each other.These regulating and controlling sequences include but not limited to targeting sequencing, poly- adenosine Polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence and transcription terminator.Bottom line, regulating and controlling sequence include promoter with And the termination signal of transcription and translation.Regulating and controlling sequence can together be carried with the connector for introducing specific restriction sites purpose For the specific restriction sites promote the connection of regulating and controlling sequence and the code area for the polynucleotides for encoding polypeptide.
It is operably connected：Mean such configuration, wherein regulating and controlling sequence is placed in the code sequence relative to polynucleotides The appropriate location of row so that regulating and controlling sequence instructs the expression of coded sequence.
Expression：Mean to include any step for being related to polypeptide generation comprising but be not limited to transcription, posttranscriptional modification, fly swiftly It translates, posttranslational modification and secretion.
Expression vector：Mean linear or cricoid DNA molecular, it is and described it includes the polynucleotides of coding polypeptide Polynucleotides are operably connected with the additional nucleotides for being used for its expression are provided.
Host cell：Mean that any cell type, the cell type include the core of polynucleotides of the present invention for using Conversion, transfection, transduction of acid con-struct or expression vector etc. are susceptible (susceptible).Term " host cell " is covered The offspring of any parental cell is different from parental cell due to the mutation occurred in a replication process.
Raw oil：Refer to (also referred to as non-degummed oil) from or mixtures thereof the expressed oil of such as plant origin or extract oil, Including but not limited to apricot kernel oil, babassu oil, 'Heijialun ' seed oil, rapeseed oil, cashew nut oil, castor oil, coconut oil, Yuan wither oily, beautiful Rice bran oil, cottonseed oil, Crambe abyssinica oil, grape seed oil, hazelnut oil, awns nut oil, galam butter, white awns flower seed oil, mustard oil, ox foot Oil, hemp-seed oil, jatropha oil, linseed oil, olive oil, palm oil, palm-kernel oil, palm olein, pine-seed oil, American pistachios Oil, rapeseed oil, rice bran oil, safflower oil, camellia caul-fat, sesame oil, peanut oil, soybean oil, sunflower seed oil, tall oil, English walnut Oil, walnut oil have the various of the fatty acid composition changed via genetically modified organism, GMO (GMO) or traditional " breeding " " natural " oil, such as high oleic acid oil, low linolenic oil or low saturated oils (high oleic acid Canola oil, low linolenic soybean oil or high hard Resin acid sunflower oil).
Degummed oil：Refer to being removed from the oil the phosphatide that can not be hydrated, the phosphatide that can be hydrated and lecithin (being referred to as " glue ") With generate can be used for food production and/or non-food applications (such as biodiesel) degummed oil or fat products after obtain Oil.
The present invention relates to not characterizing from Labilithrix luteola bacterium novel, to be labeled as substrate extensive The polypeptide of phospholipase C carries out rite-directed mutagenesis, for aforementioned polypeptides, from its use in degumming or other application is not described On the way.
An embodiment according to the present invention, the inventors discovered that, oil is carried out to utilize in the method for degumming The extensive substrate specificity of the mutant and high activity, strong acid resistance, the feature of strong heat resistance obtain highly effective phosphatide Hydrolysis.
Specifically, the present invention relates to the mutant of the phospholipase C with PC, PE and PI- activity specific, for phosphorus Phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE) and phosphatidylinositols (PI) are active.
Production method
Be labeled as using in Labilithrix luteola bacterial strains the gene of extensive substrate specificity phospholipase C polypeptide as Template (amino acid sequence such as SEQ ID No：Shown in 6, GeneBank accession number is：AKU93446.1), nucleotide primer is designed, It is expanded by PCR method and obtains the phospholipase C mutant.Then, the specific phospholipase C mutant genes obtained are inserted into Into suitable expression vector, to generate the recombinant vector containing specific phospholipase C mutant genes, and will be described Recombinant vector is transformed into suitable host cell.The microorganism of the conversion is subjected to shaking flask culture, or in laboratory or work It is thin to cultivate that small-scale or large scale fermentation (including continuous, in batches, batch feeding or solid state fermentation) is carried out in industry fermentation tank Born of the same parents.The culture is occurred in suitable nutrient medium using known program in this field, the culture medium include carbon source and Nitrogen source and inorganic salts.Suitable culture medium can obtain from commercial supplier or can be according to disclosed composition (for example, in U.S.'s allusion quotation In the catalogue of type culture collection) it prepares.It, can be directly from culture medium if polypeptide is secreted into the nutrient medium Middle recycling polypeptide.If polypeptide is not secreted, can be recycled from cell pyrolysis liquid.
Polypeptide or protein with phospholipase C activity can be detected using known method in this field, referring to following " activity of phospholipase measurement " part.
Polypeptide can use the known method in this field to recycle.For example, the polypeptide can be by conventional program, including but not It is limited to, collects, centrifuges, filters, extracts, is spray-dried, evaporates or precipitates, from the nutrient medium because receiving.On the one hand, it recycles Include the zymotic fluid of the polypeptide.
The polypeptide can be purified by known multiple programs in this field to obtain substantially pure polypeptide, these journeys Sequence includes but not limited to:Chromatography, electrophoretic procedures, differential solubilities (for example, ammonium sulfate precipitation), SDS-PAGE or extraction (ginseng See for example, protein purification (Protein Purification), Jansen (Janson) and relies and step on (Ryden) editor, VCH publishing houses (VCH Publishers), New York, 1989).
In an alternative aspect, which is not recovered, but uses the host for the present invention for expressing the polypeptide thin Born of the same parents are as the polypeptide source.
Activity of phospholipase measures
The present invention provides any combination of separation of the phospholipase C mutant with PC, PE and PI- activity specific, The polypeptide (for example, enzyme, antibody) of synthesis or recombination, and encode their nucleic acid.If polypeptide have activity of phospholipase and In the scope of the present invention, then can be determined using any one of known many activity of phospholipase measurement in this field. For determining that the conventional scheme of phospholipase A, B, D and C is well known in the art.
Exemplary active measurement includes turbidimetric analysis turbidimetry, methylumbelliferyl ketone group phosphocholine (fluorescence) measurement, the luxs A Pu Red (fluorescence) phosphatide enzymatic determination, thin-layer chromatography measure (TLC), cell dissolution measures and p-nitrophenyl phosphocholine measures.Make With these measurement, polypeptide, peptide or antibody can be quickly screened for activity of phospholipase.In addition the instance section of the application describes Measurement, such as31P-NMR。
In a preferred embodiment, PC, PE and PI- specific phospholipase C mutant of the invention can be reduced slightly PC, PE and PI content in liquefaction.Preferably, when being applied in 10mg zymoproteins/kg oil under the most suitable pH in the mutant When, phospholipase C mutant of the invention can respectively reduce at least 35% of PC, PE and PI content in raw oil, more preferably At least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
In another embodiment, the most suitable pH ranges of phospholipase C mutant of the present invention are between 4.0 to 7.5, More preferably from 4.5 to 6.0.
In a further embodiment, it can be reduced with the active mutant of PC, PE and PI- specific phospholipase C crude The phosphatidyl-ethanolamine of soybean oil, 55% or more of phosphatidyl choline and/or phosphatidylinositol content, such as 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, phosphatidyl-ethanolamine, phosphatidyl choline and/or phosphatidylinositols contain The reduction of amount be addition 100mg zymoproteins (EP)/kg oil and at 50 DEG C after pH is incubated oil and enzyme 2 hours for 5.5 time it is logical It crosses31What P-NMR was determined.
Including incubating in the low Aquo System of 3% water based on oil mass at 50 DEG C -60 DEG C again in other embodiment 4mg zymoproteins/kg oil is educated after 5 hours, if being harmonious measured by plasma emlssion spectrometry (I CP-OES) by inductance, tool There is the active mutant of PC, PE and PI- specific phospholipase C that can reduce the phosphorus content of crude soybean oil to 15mg/kg oil Or it is less.
For specifically, the ability which reduces phosphorus content can be determined using crude soy oil, this is thick Soya-bean oil processed includes as 80-140ppm phosphorus existing for phosphatidic acid (PA), as 140- existing for phosphatidyl-ethanolamine (PE) 200ppm phosphorus, as 70-110ppm phosphorus existing for phosphatidylinositols (pI) and as 130- existing for phosphatidyl choline (PC) 200ppm phosphorus:Phosphorus content passes through31P-NMR is measured.
Specifically, the reduction of phosphorus content and/or phosphatidyl-ethanolamine and/or phosphatidyl choline and/or phosphatidylinositols contain The reduction of amount can obtain in oil degumming methods, and this approach includes the following steps:
I) optionally, by handling crude soybean oil with acid/base as follows：With corresponding to 0.05% based on oil mass The amount of (100% pure orthophosphoric acid) adds 85% orthophosphoric acid solution, is mixed 5 minutes in ultrasonic bath, then in circulator It is incubated 15 minutes and in ultrasonic bath to be divided using 4M NaOH alkali neutralizations 5 with pure phosphoric acid a great deal of (from 0.5 to 0.15) Clock；
Ii including) being added to the oil with the amount of 4mg zymoproteins/kg oil in the low Aquo System of 3% water based on oil mass The phospholipase C mutant, and the oil and the phospholipase C mutant is made to be subjected to ultrasonication 5 minutes；
Iii) in the case where being stirred with 20rpm, be incubated at 50 DEG C -60 DEG C the polypeptide and oil 5 hours；
Iv the oil and the phospholipase C mutant 15 minutes) are centrifuged at 85 DEG C at 700g.
Usually with parts per million by phospholipid measurement in oil be " phosphorus content ".Table 1 lists phosphorus present in main oilseed crops The typical amount of fat and each functional group are with the distribution of the percentages of phosphatide present in oil.
Table 1:The typical level and phosphatide of common oilseeds are distributed
Soya-bean oil Canola oil Sunflower oil
Phosphorus (ppm) 400-1500 200-900 300-700
PC% 12-46 25-40 29-52
PE% 8-34 15-25 17-26
PA% 17-26 10-20 15-30
PI% 2-15 2-25 11-22
The more complete degumming of high phosphorus oil can be realized using the enzyme of the present invention and method, for example, with more than 200ppm Phosphorus oil, preferably more than 300ppm, 400ppm, 500ppm, 600ppm, 700ppm, 800ppm, 900ppm are even more excellent The selection of land oil comprises more than 1100ppm phosphorus.
Preferably, which includes phosphatidyl choline (PC), phosphatidyl-ethanolamine (PE) and phosphatidylinositols (pI).It is preferred that Ground, the oil include from phosphatidylinositols (pI) be more than 50ppm phosphorus, more preferably it comprise more than 80ppm, 110ppm, 130ppm PI, even more preferably it comprise more than 160ppm, preferably it include from PI is more than 180ppm phosphorus.It is preferred that Ground, the oil include from phosphatidyl choline (PC) be more than 100ppm phosphorus, more preferably it comprise more than 160ppm, 210ppm, 260ppm PC, even more preferably it comprise more than 310ppm, preferably it include from PC is more than 410ppm phosphorus.It is preferred that Ground, the oil include from phosphatidyl-ethanolamine (PE) be more than 80ppm phosphorus, more preferably it comprise more than 110ppm, 130ppm, 160ppmPE, even more preferably it includes super 210ppm, and it is more than 310ppm phosphorus that preferably it, which includes from PE,.
In a preferred embodiment, which is edible oil.It is highly preferred that the edible oil is selected from rice bran, rapeseed, palm fibre Palmitic acid, peanut and other perpendicular fruit, soybean, corn, rape and sunflower oils.The phosphatidase of the present invention can be used for any " degumming " mistake Journey, including water degumming, ALCON degumming of oils (for example, be used for soybean), safinco degummings, " overdegum ", UF degummings, TOP are de- Glue, single degumming, dry degumming and ENZYMAXTMDegumming.
In the following example, the phospholipase C mutant of the present invention referred to is that amino acid sequence is SEQ ID：Shown in 2 Sequence.
Embodiment 1：Clone and expression
Coding phosphatidase gene be cloned from the bacterial strain indicated above shown by routine techniques or conduct Synthetic gene is ordered and is inserted into suitable plasmid.
Selection one is with the clone of correct recombination sequence and by homologous recombination that corresponding plasmid pMA5 is whole The gene construct is expressed in closing into B. subtilis host cell genome.
Kanamycin resistant transformants are analyzed by PCR, to verify the correct size of amplified fragments.
Embodiment 2：Phospholipase C purifies
It usesThe packed bed of G-25 resins, by cell free broth buffer-exchanged to 50mM MES pH6.5.The fraction of collection is loaded on Source 15S cation exchangers, and use 0-100%50mM in 10CV The gradient of MES+0.5M NaCl pH 6.5 is eluted.Fraction is analyzed by SDS-PAGE (reducing condition), and based on pure Degree is collected.
Embodiment 3：Rite-directed mutagenesis
Using the phospholipase C gene sequence of the recombined bacillus subtilis of the expression construct comprising integration as template, design The 42nd Aspartic acid mutations of phospholipase C are successively glutamic acid, the 179th leucine are sported alanine by mutant primer, Phospholipase C mutant is obtained.Conical flask of the correct mutant from plating to 500mL will finally be sequenced (Erlenmeyer flask) on shaking table in cultivated, each conical flask includes cultures of the 100ml based on yeast extract Base.This is cloned at 30 DEG C and is cultivated 2 days.Harvest comprising supernatant enzyme and as described in above-described embodiment 2 by the enzyme into Row purifying.
Introducing the rite-directed mutagenesis primer that D42E is mutated is：
Forward primer 5 '-GCGCACTGGGAAGCCCAGAGCGTGACGAACG-3 '
Reverse primer 5 '-CACGCTCTGGGCTTCCCAGTGCGCTTCCTGGTT-3 '
Introducing the rite-directed mutagenesis primer that L179A is mutated is：
Forward primer 5 '-CTCGGACTCGCAGCGCACTACTTCACCGATCTGACG-3 '
Reverse primer 5 '-GTGAAGTAGTGCGCTGCGAGTCCGAGCTCGTAGCAG-3 '
Embodiment 4：Specificity of the phospholipase C mutant to PC, PE, PI, PA of the PLC of purifying
It uses31P-NMR determine the present invention phospholipase C enzyme andSubstrate specificity.This measurement follows The conversion of each phosphatide shown in Fig. 1 in oil environment, and the substrate specificity and preference of phosphatidase are disclosed, and carry The instruction of the Optimal pH of enzyme is supplied.
Substrate
Use the crude soybean oil of the phosphatide of the specificity with the following content measured by P-NMR.
PA:80-140ppm phosphorus (P)
PE:140-200ppm P
PI:70-110ppm P
PC:130-200ppm P
Other raw oils are also used in the measurement, for example, from rapeseed, sunflower, corn, cottonseed, peanut, rice The raw oil of chaff.Primary standard is that the oil includes each specific phospholipase (being significantly higher than NMR quantitative limits) of minimum 30ppm.It is moving Ensure to mix (it is precipitated with the time) before liquid raw oil.
Buffer solution and enzyme
By 7.5 solution of 0.2M Cs-EDTA pH:EDTA (5.85g) disperses in MQ- water (50mL).Use 50%w/w CsOH (about 30mL) adjusts pH to 7.5, will be completely dissolved EDTA.MQ- water is added to the total volume of 100mL, to provide The concentration of 0.2M.
Internal standard:2mg/mL triphenyl phosphates (TPP) solution in MeOH.
PH buffer solutions:
100mM sodium acetates pH 4.0
100mM sodium acetates pH 5.5
100mM sodium acetates pH 7.0
Enzyme:It is diluted to the concentration of 0.9,0.27 and 0.09mg zymoproteins (EP)/mL in three kinds of buffer solutions, and keeps cold But the same day uses.
It measures
250 microlitres of raw oil is weighed in 2mL Eppendorf tubes, and adds and is diluted in desired pH buffer solutions 25 microlitres of enzymes.This generates 10,30 and 100mg Ep/kg oil.At 50 DEG C, which is incubated 2h in heating shaking table. Then addition 0.500mL phosphate standards solution, 0.5mL chloroforms d (CDC13) and 0.5mL Cs-EDTA buffer solutions.It shakes in 30s Then centrifugation (desk centrifuge, 4min, 13,000rpm) is swung to be separated afterwards.Lower phase is transferred in NMR- pipes.With 128 Secondary scanning, delay operation in 5 seconds31P-NMR.Integrate all signals.Assignment (at 25 DEG C, ppm is about worth):1.7(PA)、-0.1 (PE)、-0.5(PI)、-0.8(PC).According to exact pH value, temperature, sample concentration etc., signal location can be substantially change. The concentration of various species is calculated with " ppm P ", that is, mg element phosphors/kg oil samples.Therefore, ppm P=I/I (I S) * n (I S) * M (P)/m (oil).Phosphoric residue is calculated with the ratio of same concentrations in the phospholipid concentration and blank sample in the sample of enzymatic treatment Fat %.As a result it is summarized in the following table 2.
Table 2：The specificity of phospholipase C
For expression activitiy, shown belowThe performance of phosphatidase.
Table 3：The specificity of phosphatidase
Concentration is estimated as 15mg/mL.
Embodiment 5：Degumming measures
Simulation commercial scale degumming degumming measure in test the present invention phospholipase C mutant andProperty Energy.After degumming, which measures following parameter in oil phase：
A) it, is examined by the high performance liquid chromatography (HPLC) or electron spray that are coupled with evaporative light scattering detector (ELSD) Survey the diacylglycerol content that device (Corona Veo) measures.
B) each phospholipid species, are quantified according to national standard GB 5009.272-2016：Phosphatidyl choline (PC)；Phosphatidylinositols (PI)；Phosphatidyl-ethanolamine (PE)；Phosphatidic acid (PA)
C), by inductance be harmonious plasma emlssion spectrometry (ICP-OES) total phosphorus reduce.
The phospholipid composite of the crude soybean oil used in experiment.With the phosphorus from each phospholipid species, surveyed by LC/MS Measure the composition.
The phospholipid composite (mg/kg phosphorus) of raw oil is as follows：
PA:295
PE:125
PI:84
PC:229
Degumming measures
Initial acid/base pretreatment (or not pre-processing) crude soybean oil (75g), to promote insoluble phosphatide salt to be converted to More hydratable form, and ensure the environment suitable for the enzyme.It is pre-processed by acid/base accomplished below, to be based on oil mass etc. Sour addition is carried out using orthophosphoric acid in the amount of 0.05% (100% pure orthophosphoric acid), and at ultrasonic bath (BRANSON3510) Middle mixing 5min, and 15min is incubated in circulator, then in ultrasonic bath for pure phosphoric acid equivalent (from 0.5 to 1.5) the 4M NaOH used carry out alkali neutralization and continue 5min.In 100ml centrifuge tubes, cylindrical type, conical bottom, in low water content System's (overall 3% water based on oil mass) carries out enzyme reaction.Sample is subjected to supersound process 5min, then selected in heating is dry It is (small from 1 to 5 that selected incubation time is incubated in the case where being stirred with 20rpm at the temperature (from 50 DEG C to 60 DEG C) selected When).In order to which the mixture is separated into oil phase and heavy water/glue phase, at 700g, these samples 15min (Ke Le are centrifuged at 85 DEG C Instrument company (Koehler Instruments), K600X2 oil centrifuges).
A) diglyceride measures
HPLC-ELSD or HPLC-Corona Veo methods (using DIONEX equipment and Lichrocart Si-60,5 μm, Lichrosphere 250-4mm, Merck column) it is the principle based on AOCS official method Cd lld-96, and quantify glycerine two Ester content is down to 0.1wt%.
B) quantitative analysis phosphatide
Each phospholipid species content is measured according to national standard GB 5009.272-2016:Phosphatidyl choline (PC)；Phosphatidyl-4 Alcohol (PI)；Phosphatide ester ethanol amine (PE) and phosphatidic acid (PA).The data are handled using 4.1 editions softwares of Mass Lynx.
C) phosphorus/phospholipid measurement
The ICP-OES quantifies phosphorus (P) content down to 4ppm with the accuracy of about+1ppm P.
Following example 5 describes to 7 and measures obtained result using the degumming of the example.
Embodiment 6：The influence of enzyme dosage
In degumming measurement, the phospholipase C mutant of the present invention has been used in raw oil with various enzyme dosages.At 60 DEG C After lower 1,2,3 and 5hr of enzymatic degumming, measuring diacylglycerol content, (NaOH with the equivalent of 0.05% phosphoric acid/1.5 is pretreated Oil).As a result it is shown in the following table 4.
Table 4：Pass through the increasing of the diglyceride after the enzymatic treatment of the HPLC-ELSD pretreated soybean oils of acid/base measured Add
In degumming measurement, phospholipase C mutant of the invention at 1-5 hour it is interior with from 1 to 20mg Ep/kg oil enzyme Dosage is by PI phospholipid conversions at diglyceride.Data confirm that dose response effect under high enzyme dosage has faster phosphatide It is converted into diglyceride.
Embodiment 7：At 50 DEG C, using phospholipase C mutant of the present invention and
In degumming measurement, at 50 DEG C, with the pretreated raw oils of equivalent NaOH of 0.05% phosphoric acid/1.5.It is de- in enzyme process After 1,2,3 and 5hr of glue, diacylglycerol content is measured.As a result it is shown in Table 5.
Table 5：As measured by HPLC-ELSD, in the enzyme of the pretreated soybean oils of equivalent NaOH of 0.05% phosphoric acid/1.5 After method processing, the increase of diglyceride.
Table 5
At 50 DEG C, using phospholipase C mutant degumming of the present invention, significant diglyceride is caused to be formed.By more DG increase find, withPLC is compared, the phosphorus content in degummed oil be reduced to it is desirable less than 5mg/kg most It is horizontal eventually, and equal to ' complete ' conversion.
Embodiment 8：At 60 DEG C, using phospholipase C mutant of the present invention and
In degumming measurement, at 60 DEG C, with the pretreated raw oils of equivalent NaOH of 0.05% phosphoric acid/1.5.It is de- in enzyme process After 1,2,3 and 5hr of glue, diacylglycerol content is measured.As a result it is shown in Table 6.
Table 6：As measured by HPLC-ELSD, in the enzyme of the pretreated soybean oils of equivalent NaOH of 0.05% phosphoric acid/1.5 After method processing, the increase of diglyceride.
At 60 DEG C withThe effect of PLC is compared, and the advantageous effect of phospholipase C mutant degumming of the present invention is bright It is aobvious, convert most phosphatide.
It is described herein and claimed invention is not limited to the ranges of particular aspects disclosed here, because of these sides Face is intended as illustrations of several aspects of the invention.It is expected that any equivalent aspect is all in the scope of the present invention.In fact, removing Except those of shown here and description, different modifications of the invention are for those of ordinary skills from foregoing description It will be apparent.Such modification, which is also intended to, to be fallen within the scope of the appended claims.In case of conflict, to include fixed Subject to the present disclosure of justice.
Sequence table
<110>The Jiangsu bio tech ltd Zhong Mei
<120>A kind of mutant of phosphatidase and its application
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 333
<212> PRT
<213>Artificial sequence
<400> 1
MALHRSLLAL LVASLASLIA CAAPSDGSDD GEEVDNQEAH WEAQSVTNES ESTHLWIVDR 60
GIDILAHHGD SDPVAARAWG LMTNATCRAQ WQQGLYDADF KAAYNNGRSD LPPNPSDVQV 120
GLAGATWASH FYDPDTGKNY KGETSPTAYS EASAHLSLAK ENHLSDGKAK GCYELGLALH 180
YFTDLTQPMH AANFTAVNRP AKLHSNLEGY SMELQERYPL EDWSGPPSGT TRDFLVKTAK 240
DSKPLFMEGV EAIVAAYKSY TGWRILQCRN IDAAAWRFVE RQHVDYRDCW EGNAGVDAVI 300
GKTLRFAQER TAQYIYLVAK EIGGDAPTTS GSP 333
<210> 2
<211> 333
<212> PRT
<213>Artificial sequence
<400> 2
MALHRSLLAL LVASLASLIA CAAPSDGSDD GEEVDNQEAH WEAQSVTNES ESTHLWIVDR 60
GIDILAHHGD SDPVAARAWG LMTNATCRAQ WQQGLYDADF KAAYNNGRSD LPPNPSDVQV 120
GLAGATWASH FYDPDTGKNY KGETSPTAYS EASAHLSLAK ENHLSDGKAK GCYELGLAAH 180
YFTDLTQPMH AANFTAVNRP AKLHSNLEGY SMELQERYPL EDWSGPPSGT TRDFLVKTAK 240
DSKPLFMEGV EAIVAAYKSY TGWRILQCRN IDAAAWRFVE RQHVDYRDCW EGNAGVDAVI 300
GKTLRFAQER TAQYIYLVAK EIGGDAPTTS GSP 333
<210> 3
<211> 333
<212> PRT
<213>Artificial sequence
<400> 3
MALHRSLLAL LVASLASLIA CAAPSDGSDD GEEVDNQEAH WEAQSVTNES ESTHLWIVDR 60
GIDILAHHGD SDPVAARAWG LMTNATCRAQ WQQGLYDADF KAAYNNGRSD LPPNPSDVVV 120
GLAGATWASH FYDPDTGKNY KGETSPTAYS EASAHLSLAK ENHLSDGKAK GCYELGLALH 180
YFTDLTQPMH AANFTAVNRP AKLHSNLEGY SMELQERYPL EDWSGPPSGT TRDFLVKTAK 240
DSKPLFMEGV EAIVAAYKSY TGWRILQCRN IDAAAWRFVE RQHVDYRDCW EGNAGVDAVI 300
GKTLRFAQER TAQYIYLVAK EIGGDAPTTS GSP 333
<210> 4
<211> 333
<212> PRT
<213>Artificial sequence
<400> 4
MALHRSLLAL LVASLASLIA CAAPSDGSDD GEEVDNQEAH WDAQSVTNES ESTHLWIVDR 60
GIDILAHHGD SDPVAARAWG LMTNATCRAQ WQQGLYDADF KAAYNNGRSD LPPNPSDVVV 120
GLAGATWASH FYDPDTGKNY KGETSPTAYS EASAHLSLAK ENHLSDGKAK GCYELGLALH 180
YFTDLTQPMH AANFTAVNRP AKLHSNLEGY SWELQERYPL EDWSGPPSGT TRDFLVKTAK 240
DSKPLFMEGV EAIVAAYKSY TGWRILQCRN IDAAAWRFVE RQHVDYRDCW EGNAGVDAVI 300
GKTLRFAQER TAQYIYLVAK EIGGDAPTTS GSP 333
<210> 5
<211> 333
<212> PRT
<213>Artificial sequence
<400> 5
MALHRSLLAL LVASLASLIA CAAPSDGSDD GEEVDNQEAH WDAQSVTNES ESTHLWIVDR 60
GIDILAHHGD SDPVAARAWG LMTNATCRAQ WQQGLYDADF KAAYNNGRSD LPPNPSDVQV 120
GLAGATWASH FYDPDTGKNY KGETSPTAYS EASAHLSLAK ENHLSDGKAK GCYELGLAAH 180
YFTDLTQPMH AANFTAVNRP AKLHSNLEGY SWELQERYPL EDWSGPPSGT TRDFLVKTAK 240
DSKPLFMEGV EAIVAAYKSY TGWRILQCRN IDAAAWRFVE RQHVDYRDCW EGNAGVDAVI 300
GKTLRFAQER TAQYIYLVAK EIGGDAPTTS GSP 333
<210> 6
<211> 333
<212> PRT
<213> Labilithrix luteola
<400> 6
MALHRSLLAL LVASLASLIA CAAPSDGSDD GEEVDNQEAH WDAQSVTNES ESTHLWIVDR 60
GIDILAHHGD SDPVAARAWG LMTNATCRAQ WQQGLYDADF KAAYNNGRSD LPPNPSDVQV 120
GLAGATWASH FYDPDTGKNY KGETSPTAYS EASAHLSLAK ENHLSDGKAK GCYELGLALH 180
YFTDLTQPMH AANFTAVNRP AKLHSNLEGY SMELQERYPL EDWSGPPSGT TRDFLVKTAK 240
DSKPLFMEGV EAIVAAYKSY TGWRILQCRN IDAAAWRFVE RQHVDYRDCW EGNAGVDAVI 300
GKTLRFAQER TAQYIYLVAK EIGGDAPTTS GSP 333
<210> 7
<211> 1002
<212> DNA
<213> Labilithrix luteola
<400> 7
atggcactgc accgttcgct gctggccctc ctcgtcgcct cgttggcctc cctcatcgcc 60
tgtgcggcac cctccgatgg cagtgacgac ggtgaggagg tcgacaacca ggaagcgcac 120
tgggccgccc agagcgtgac gaacgagtcg gagtcgacgc acctctggat cgtcgaccga 180
ggcatcgaca ttctcgccca tcacggcgac tcggatccgg tcgctgcgcg tgcgtggggt 240
ctcatgacca acgcaacgtg ccgcgctcag tggcagcagg gcctctacga tgccgacttc 300
aaggcggcct acaacaacgg gcggtcggac cttccgccga atccgtccga cgttcaggtc 360
ggtctggccg gcgcgacctg ggcgagtcac ttctacgatc cggacacggg gaagaactac 420
aagggcgaga cgagccctac ggcttacagc gaggcctcgg cacacctctc gctcgccaag 480
gagaatcacc tctccgacgg caaggccaag ggctgctacg agctcggact cgcactgcac 540
tacttcaccg atctgacgca gccgatgcat gccgcgaact tcacggcggt caatcgtccg 600
gccaagctgc attcgaatct cgagggatat tcgatggagc tgcaagaacg atatccgctc 660
gaagactgga gcggaccgcc gagcggcacg acacgcgatt tcctcgtcaa aaccgcgaaa 720
gactccaagc cgctcttcat ggaaggcgtc gaggccatcg tcgcggccta caagtcctat 780
accggctggc gcattctcca gtgtcgcaac atcgatgcag ccgcgtggcg gttcgtcgag 840
cgccagcacg tcgattatcg cgattgttgg gagggtaatg cgggcgtcga tgccgtcatc 900
ggcaaaacgc tccggttcgc gcaggagcgg acggcccaat acatctatct cgtcgccaag 960
gagattgggg gcgacgcccc gacgacttcg ggatctccct ga 1002
<210> 8
<211> 1002
<212> DNA
<213>Artificial sequence
<400> 8
atggcactgc accgttcgct gctggccctc ctcgtcgcct cgttggcctc cctcatcgcc 60
tgtgcggcac cctccgatgg cagtgacgac ggtgaggagg tcgacaacca ggaagcgcac 120
tgggaagccc agagcgtgac gaacgagtcg gagtcgacgc acctctggat cgtcgaccga 180
ggcatcgaca ttctcgccca tcacggcgac tcggatccgg tcgctgcgcg tgcgtggggt 240
ctcatgacca acgcaacgtg ccgcgctcag tggcagcagg gcctctacga tgccgacttc 300
aaggcggcct acaacaacgg gcggtcggac cttccgccga atccgtccga cgttcaggtc 360
ggtctggccg gcgcgacctg ggcgagtcac ttctacgatc cggacacggg gaagaactac 420
aagggcgaga cgagccctac ggcttacagc gaggcctcgg cacacctctc gctcgccaag 480
gagaatcacc tctccgacgg caaggccaag ggctgctacg agctcggact cgcactgcac 540
tacttcaccg atctgacgca gccgatgcat gccgcgaact tcacggcggt caatcgtccg 600
gccaagctgc attcgaatct cgagggatat tcgatggagc tgcaagaacg atatccgctc 660
gaagactgga gcggaccgcc gagcggcacg acacgcgatt tcctcgtcaa aaccgcgaaa 720
gactccaagc cgctcttcat ggaaggcgtc gaggccatcg tcgcggccta caagtcctat 780
accggctggc gcattctcca gtgtcgcaac atcgatgcag ccgcgtggcg gttcgtcgag 840
cgccagcacg tcgattatcg cgattgttgg gagggtaatg cgggcgtcga tgccgtcatc 900
ggcaaaacgc tccggttcgc gcaggagcgg acggcccaat acatctatct cgtcgccaag 960
gagattgggg gcgacgcccc gacgacttcg ggatctccct ga 1002
<210> 9
<211> 1002
<212> DNA
<213>Artificial sequence
<400> 9
atggcactgc accgttcgct gctggccctc ctcgtcgcct cgttggcctc cctcatcgcc 60
tgtgcggcac cctccgatgg cagtgacgac ggtgaggagg tcgacaacca ggaagcgcac 120
tgggaagccc agagcgtgac gaacgagtcg gagtcgacgc acctctggat cgtcgaccga 180
ggcatcgaca ttctcgccca tcacggcgac tcggatccgg tcgctgcgcg tgcgtggggt 240
ctcatgacca acgcaacgtg ccgcgctcag tggcagcagg gcctctacga tgccgacttc 300
aaggcggcct acaacaacgg gcggtcggac cttccgccga atccgtccga cgttcaggtc 360
ggtctggccg gcgcgacctg ggcgagtcac ttctacgatc cggacacggg gaagaactac 420
aagggcgaga cgagccctac ggcttacagc gaggcctcgg cacacctctc gctcgccaag 480
gagaatcacc tctccgacgg caaggccaag ggctgctacg agctcggact cgcagcgcac 540
tacttcaccg atctgacgca gccgatgcat gccgcgaact tcacggcggt caatcgtccg 600
gccaagctgc attcgaatct cgagggatat tcgatggagc tgcaagaacg atatccgctc 660
gaagactgga gcggaccgcc gagcggcacg acacgcgatt tcctcgtcaa aaccgcgaaa 720
gactccaagc cgctcttcat ggaaggcgtc gaggccatcg tcgcggccta caagtcctat 780
accggctggc gcattctcca gtgtcgcaac atcgatgcag ccgcgtggcg gttcgtcgag 840
cgccagcacg tcgattatcg cgattgttgg gagggtaatg cgggcgtcga tgccgtcatc 900
ggcaaaacgc tccggttcgc gcaggagcgg acggcccaat acatctatct cgtcgccaag 960
gagattgggg gcgacgcccc gacgacttcg ggatctccct ga 1002
<210> 10
<211> 1002
<212> DNA
<213>Artificial sequence
<400> 10
atggcactgc accgttcgct gctggccctc ctcgtcgcct cgttggcctc cctcatcgcc 60
tgtgcggcac cctccgatgg cagtgacgac ggtgaggagg tcgacaacca ggaagcgcac 120
tgggaagccc agagcgtgac gaacgagtcg gagtcgacgc acctctggat cgtcgaccga 180
ggcatcgaca ttctcgccca tcacggcgac tcggatccgg tcgctgcgcg tgcgtggggt 240
ctcatgacca acgcaacgtg ccgcgctcag tggcagcagg gcctctacga tgccgacttc 300
aaggcggcct acaacaacgg gcggtcggac cttccgccga atccgtccga cgttgtggtc 360
ggtctggccg gcgcgacctg ggcgagtcac ttctacgatc cggacacggg gaagaactac 420
aagggcgaga cgagccctac ggcttacagc gaggcctcgg cacacctctc gctcgccaag 480
gagaatcacc tctccgacgg caaggccaag ggctgctacg agctcggact cgcactgcac 540
tacttcaccg atctgacgca gccgatgcat gccgcgaact tcacggcggt caatcgtccg 600
gccaagctgc attcgaatct cgagggatat tcgatggagc tgcaagaacg atatccgctc 660
gaagactgga gcggaccgcc gagcggcacg acacgcgatt tcctcgtcaa aaccgcgaaa 720
gactccaagc cgctcttcat ggaaggcgtc gaggccatcg tcgcggccta caagtcctat 780
accggctggc gcattctcca gtgtcgcaac atcgatgcag ccgcgtggcg gttcgtcgag 840
cgccagcacg tcgattatcg cgattgttgg gagggtaatg cgggcgtcga tgccgtcatc 900
ggcaaaacgc tccggttcgc gcaggagcgg acggcccaat acatctatct cgtcgccaag 960
gagattgggg gcgacgcccc gacgacttcg ggatctccct ga 1002
<210> 11
<211> 1002
<212> DNA
<213>Artificial sequence
<400> 11
atggcactgc accgttcgct gctggccctc ctcgtcgcct cgttggcctc cctcatcgcc 60
tgtgcggcac cctccgatgg cagtgacgac ggtgaggagg tcgacaacca ggaagcgcac 120
tgggacgccc agagcgtgac gaacgagtcg gagtcgacgc acctctggat cgtcgaccga 180
ggcatcgaca ttctcgccca tcacggcgac tcggatccgg tcgctgcgcg tgcgtggggt 240
ctcatgacca acgcaacgtg ccgcgctcag tggcagcagg gcctctacga tgccgacttc 300
aaggcggcct acaacaacgg gcggtcggac cttccgccga atccgtccga cgttgtggtc 360
ggtctggccg gcgcgacctg ggcgagtcac ttctacgatc cggacacggg gaagaactac 420
aagggcgaga cgagccctac ggcttacagc gaggcctcgg cacacctctc gctcgccaag 480
gagaatcacc tctccgacgg caaggccaag ggctgctacg agctcggact cgcactgcac 540
tacttcaccg atctgacgca gccgatgcat gccgcgaact tcacggcggt caatcgtccg 600
gccaagctgc attcgaatct cgagggatat tcgtgggagc tgcaagaacg atatccgctc 660
gaagactgga gcggaccgcc gagcggcacg acacgcgatt tcctcgtcaa aaccgcgaaa 720
gactccaagc cgctcttcat ggaaggcgtc gaggccatcg tcgcggccta caagtcctat 780
accggctggc gcattctcca gtgtcgcaac atcgatgcag ccgcgtggcg gttcgtcgag 840
cgccagcacg tcgattatcg cgattgttgg gagggtaatg cgggcgtcga tgccgtcatc 900
ggcaaaacgc tccggttcgc gcaggagcgg acggcccaat acatctatct cgtcgccaag 960
gagattgggg gcgacgcccc gacgacttcg ggatctccct ga 1002
<210> 12
<211> 1002
<212> DNA
<213>Artificial sequence
<400> 12
atggcactgc accgttcgct gctggccctc ctcgtcgcct cgttggcctc cctcatcgcc 60
tgtgcggcac cctccgatgg cagtgacgac ggtgaggagg tcgacaacca ggaagcgcac 120
tgggacgccc agagcgtgac gaacgagtcg gagtcgacgc acctctggat cgtcgaccga 180
ggcatcgaca ttctcgccca tcacggcgac tcggatccgg tcgctgcgcg tgcgtggggt 240
ctcatgacca acgcaacgtg ccgcgctcag tggcagcagg gcctctacga tgccgacttc 300
aaggcggcct acaacaacgg gcggtcggac cttccgccga atccgtccga cgttcaggtc 360
ggtctggccg gcgcgacctg ggcgagtcac ttctacgatc cggacacggg gaagaactac 420
aagggcgaga cgagccctac ggcttacagc gaggcctcgg cacacctctc gctcgccaag 480
gagaatcacc tctccgacgg caaggccaag ggctgctacg agctcggact cgcagcgcac 540
tacttcaccg atctgacgca gccgatgcat gccgcgaact tcacggcggt caatcgtccg 600
gccaagctgc attcgaatct cgagggatat tcgtgggagc tgcaagaacg atatccgctc 660
gaagactgga gcggaccgcc gagcggcacg acacgcgatt tcctcgtcaa aaccgcgaaa 720
gactccaagc cgctcttcat ggaaggcgtc gaggccatcg tcgcggccta caagtcctat 780
accggctggc gcattctcca gtgtcgcaac atcgatgcag ccgcgtggcg gttcgtcgag 840
cgccagcacg tcgattatcg cgattgttgg gagggtaatg cgggcgtcga tgccgtcatc 900
ggcaaaacgc tccggttcgc gcaggagcgg acggcccaat acatctatct cgtcgccaag 960
gagattgggg gcgacgcccc gacgacttcg ggatctccct ga 1002
<210> 13
<211> 31
<212> DAN
<213>Artificial sequence
<400> 13
gcgcactggg aagcccagag cgtgacgaac g 31
<210> 14
<211> 33
<212> DAN
<213>Artificial sequence
<400> 14
cacgctctgg gcttcccagt gcgcttcctg gtt 33
<210> 15
<211> 36
<212> DAN
<213>Artificial sequence
<400> 15
ctcggactcg cagcgcacta cttcaccgat ctgacg 36
<210> 16
<211> 36
<212> DAN
<213>Artificial sequence
<400> 16
gtgaagtagt gcgctgcgag tccgagctcg tagcag 36

Claims (11)

1. the mutant of a kind of phospholipase C mutant, the phospholipase C is the phosphatidase with PC, PE and PI- activity specific C mutant contains selected from one of amino acid sequence as follows：
(a)、SEQ ID No：1 to SEQ ID No：Sequence shown in 5；
(b), with sequence shown in (a) at least 90% homogeneity and with the sequence of improved phospholipase C activity；
(c), the sequence in (a) obtains having improved phosphorus through missing, insertion and/or the one or more amino acid residues of substitution The sequence of phospholipase C activity；
Wherein sequence shown in (b) is not SEQ ID No：Sequence shown in 6.
2. phospholipase C mutant according to claim 1, it is characterised in that：Contain such as SEQ ID No：1,2,3,4 or 5 Shown in amino acid sequence.
3. phospholipase C mutant according to claim 1 or 2, it is characterised in that：Contain such as SEQ ID No：Shown in 2 Amino acid sequence.
4. phospholipase C mutant according to claim 1 or 2, it is characterised in that：The core of the phospholipase C mutant Thuja acid coded sequence contains sequence as follows：
(a)、SEQ ID No：Sequence shown in 8-12；
(b), there is at least 90% homogeneity with the sequence described in (a) and polypeptide of the coding with improved phospholipase C activity or The sequence of protein；
(c), hybridize under high stringency conditions with the sequence described in (a) and encode the polypeptide with improved phospholipase C activity Or the sequence of protein；
Wherein, the nucleotide coding sequence does not contain SEQ ID No：Sequence shown in 7.
5. a kind of nucleic acid construct or expression vector, it includes the polynucleotides of polypeptide or protein described in claim 4, institutes It states polynucleotides and is operably connected to one or more regulating and controlling sequences, the regulating and controlling sequence instructs the polypeptide or protein to exist Generation in expression vector.
6. a kind of recombinant host cell, it includes the polynucleotides of claim 4, the polynucleotides are operably connected to one A or multiple regulating and controlling sequences, the regulating and controlling sequence instruct the generation of the polypeptide or protein.
7. a kind of method generating polypeptide or protein with phospholipase C activity, it is characterised in that：The method includes：
(a), the host cell of claim 6 is cultivated under conditions of contributing to the generation of the polypeptide or protein；With
(b), the polypeptide or protein are recycled.
8. polypeptide as described in claim 7 or protein is in the method for reducing content of phospholipid in a kind of fluid composition, This method includes：
（a）A kind of fluid composition for including a certain amount of phosphatide is provided,
（b）Under conditions of being enough that the enzyme is made to react with the phosphatide, have PC, PE and PI special the fluid composition and one kind Property the contact of active phospholipase C mutant, to generate diglyceride and phosphate；And
（c）The phosphate is detached from the fluid composition.
9. according to the method described in claim 8, it is characterized in that：The described oil is a kind of edible oil.
10. the method according to any one of claim 7 to 9, it is characterised in that：It is de- that the described oil is selected from raw oil, water Glue oil, causticity refined oil and sour degummed oil.
11. method according to any one of claims 7 to 10, it is characterised in that：The described oil include phosphatidyl choline, Phosphatidyl-ethanolamine and phosphatidylinositols.
CN201810166718.6A 2018-02-28 2018-02-28 A kind of mutant of phospholipase C and its application Pending CN108384768A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810166718.6A CN108384768A (en) 2018-02-28 2018-02-28 A kind of mutant of phospholipase C and its application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810166718.6A CN108384768A (en) 2018-02-28 2018-02-28 A kind of mutant of phospholipase C and its application

Publications (1)

Publication Number Publication Date
CN108384768A true CN108384768A (en) 2018-08-10

Family

ID=63068986

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810166718.6A Pending CN108384768A (en) 2018-02-28 2018-02-28 A kind of mutant of phospholipase C and its application

Country Status (1)

Country Link
CN (1) CN108384768A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020135657A1 (en) * 2018-12-28 2020-07-02 丰益(上海)生物技术研发中心有限公司 Phospholipase c mutant with high enzyme activity
CN112522233A (en) * 2020-12-11 2021-03-19 中国农业大学 Aspergillus oryzae phospholipase C and encoding gene and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1354049A2 (en) * 2000-07-17 2003-10-22 Millenium Pharmaceuticals, Inc. 16816 and 16839, human phospholipase c molecules and uses therefor
CN101426918A (en) * 2004-03-08 2009-05-06 戴弗萨公司 Phospholipases, nucleic acids encoding them and methods for making and using them
CN102174546A (en) * 2002-04-19 2011-09-07 维莱尼姆公司 Phospholipases, nucleic acids encoding them and methods for making and using them
CN102712671A (en) * 2009-10-16 2012-10-03 帝斯曼知识产权资产管理有限公司 Phospholipases, nucleic acids encoding them and methods for making and using them
CN103314091A (en) * 2010-11-12 2013-09-18 诺维信公司 Polypeptides having phospholipase c activity and polynucleotides encoding same
CN106459934A (en) * 2014-05-15 2017-02-22 诺维信公司 Compositions comprising polypeptides having phospholipase c activity and use thereof
CN106884009A (en) * 2015-12-16 2017-06-23 丰益(上海)生物技术研发中心有限公司 The efficient phospholipase C mutant for being independent of zinc ion

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1354049A2 (en) * 2000-07-17 2003-10-22 Millenium Pharmaceuticals, Inc. 16816 and 16839, human phospholipase c molecules and uses therefor
CN102174546A (en) * 2002-04-19 2011-09-07 维莱尼姆公司 Phospholipases, nucleic acids encoding them and methods for making and using them
CN101426918A (en) * 2004-03-08 2009-05-06 戴弗萨公司 Phospholipases, nucleic acids encoding them and methods for making and using them
CN102712671A (en) * 2009-10-16 2012-10-03 帝斯曼知识产权资产管理有限公司 Phospholipases, nucleic acids encoding them and methods for making and using them
CN103314091A (en) * 2010-11-12 2013-09-18 诺维信公司 Polypeptides having phospholipase c activity and polynucleotides encoding same
CN106459934A (en) * 2014-05-15 2017-02-22 诺维信公司 Compositions comprising polypeptides having phospholipase c activity and use thereof
CN106884009A (en) * 2015-12-16 2017-06-23 丰益(上海)生物技术研发中心有限公司 The efficient phospholipase C mutant for being independent of zinc ion

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020135657A1 (en) * 2018-12-28 2020-07-02 丰益(上海)生物技术研发中心有限公司 Phospholipase c mutant with high enzyme activity
CN112522233A (en) * 2020-12-11 2021-03-19 中国农业大学 Aspergillus oryzae phospholipase C and encoding gene and application thereof
CN112522233B (en) * 2020-12-11 2022-05-03 中国农业大学 Aspergillus oryzae phospholipase C and encoding gene and application thereof

Similar Documents

Publication Publication Date Title
US9649368B2 (en) Genetically engineered microbial strains including prototheca lipid pathway genes
US10246641B2 (en) Processes for producing hydrocarbon products
US10925293B2 (en) Methods of producing lipids
EP3778866A1 (en) Recombinant yeast strain for producing nervonic acids and application thereof
CN102686737B (en) Use the manufacture method of the lipid containing lipid acid of thioesterase variant
CN102884189B (en) Thioesterase and use its lipid acid or the manufacture method of lipid
AU2009240794A1 (en) Polypeptides and methods for producing triacylglycerols comprising modified fatty acids
Ding et al. Long-chain acyl-CoA synthetase 2 is involved in seed oil production in Brassica napus
CN110114456A (en) Novel acyltransferase, variant thioesterase and its purposes
CN106103704A (en) There are the polypeptide of phospholipase C activity and the polynucleotides of these polypeptide of coding
CN108384768A (en) A kind of mutant of phospholipase C and its application
US10457890B2 (en) Phospholipase C
CN106459935A (en) Polypeptides having phospholipase c activity and polynucleotides encoding same
Xin et al. Homologous Overexpression of Diacylglycerol Acyltransferase in Oleaginous Fungus Mucor circinelloides WJ11 Enhances Lipid Accumulation under Static Solid Cultivation
Val et al. Sustainable refining of vegetable oil made easy with a designer phospholipase C enzyme
Yu et al. Transcriptomic analysis reveals key genes involved in oil accumulation and unsaturated fatty acids biosynthesis in Hibiseu manihot seeds
WO2023212726A2 (en) Regiospecific incorporation of fatty acids in triglyceride oil
Yu et al. Transcriptome sequencing and identification of key genes in the oil content accumulation and unsaturated fatty acid biosynthesis pathway of Hibiseu manihot seeds
WO2023135541A1 (en) Mutated phospholipase c enzyme
Shang et al. Cloning and expression analysis of a lipase gene from Dunaliella parva
EP4334467A1 (en) Enzymatic treatment of feedstock for hydrotreated vegetable oil (hvo) production
US20230143841A1 (en) Beta-ketoacyl-acp synthase iv variants
AU2013204308A1 (en) Methods of producing lipids
NZ627107B2 (en) Processes for producing lipids

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20180810