CN108359654A - A kind of mutant and its application with phospholipase B activity - Google Patents

A kind of mutant and its application with phospholipase B activity Download PDF

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CN108359654A
CN108359654A CN201810173368.6A CN201810173368A CN108359654A CN 108359654 A CN108359654 A CN 108359654A CN 201810173368 A CN201810173368 A CN 201810173368A CN 108359654 A CN108359654 A CN 108359654A
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sequence
phospholipase
activity
protein
polypeptide
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严明
陈圣
章志林
魏淼
陈晶晶
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Nanjing New Enzyme Biological Technology Co Ltd
Jiangsu Zhongmei Biological Technology Co Ltd
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Nanjing New Enzyme Biological Technology Co Ltd
Jiangsu Zhongmei Biological Technology Co Ltd
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Abstract

The present invention relates to a kind of mutant with phospholipase B activity and its applications.The mutant with phospholipase B activity being improved using rite-directed mutagenesis, can be effectively used for preparing choline glycerophosphatide.The present invention is also related to include constructs, carrier and the host cell of the polynucleotides and the polynucleotides for encoding the mutant enzyme, together with the method for producing the mutant and using the mutant.

Description

A kind of mutant and its application with phospholipase B activity
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 a kind of mutant with phospholipase B and its application, specifically, It is to be related to a kind of mutant with phospholipase B activity and its preparation applied to choline glycerophosphatide.
Background technology
Phosphatide is extensive in distributed in nature, all contains phosphatide in all cells, phosphatide be biomembrane basic composition at Point.Phosphatide plays metabolism and structure formation in life process, is important living matter.Phosphatidase is hydrolytic phosphatide Ester bond enzyme, be widely present in eucaryote and prokaryotes, influence the formation and again of the catabolism, biomembrane of phosphatide Group, and phosphatidase is directed to the cascade of signal.According to its action site difference, phosphatidase is divided into phospholipase A1 、A2 、B 、C , D etc..Wherein phospholipase A1, phospholipase A2It can special hydrolytic phosphatide glycerine Sn-1Or Sn-2Acyl group on position generates lysophosphatide And free fatty, to remove non-hydratable phospholipid.Phospholipase B（PLB）With hydrolase and lysophospholipase-transacylase The activity of activity, hydrolase can hydrolyze S simultaneouslyn-1And Sn-2 Position acyl group, removes the aliphatic acid in phosphatide and lysophosphatide, turns acyl Free fatty is then transferred to lysophosphatide and generates phosphatide by base enzymatic activity.
Phosphatidase includes in the industry the modification of the phospholipid emulsifier applied to food and non-food stuff there are many application, is increased The emulsification of oiling/aqueous mixtures；The enzymatic degumming that phosphatidase can be used in vegetable oil process；Starch hydrolysate（Especially It is the hydrolysate of wheaten starch）Subsequent processing to improve filtering transparency.
Choline glycerophosphatide (L-alpha glycerylphosphorylcholine, L- α-GPC) by choline, glycerine and Phosphate forms, be synthesize acetylcholine neurotransmitter precursor and all body cells in important nutrient.Phosphoglycerol courage The clinical research of alkali, which has shown it, in terms of improving neurology brain function and cognitive performance there are important medical applications to be worth, can For treating such as Alzheimer's disease, cerebellar ataxia, schizophrenia and bipolar disorder, but with regard to conduct L- α-the GPC of higher degree are needed for medicinal raw material and correlative study.Unfortunately the content of natural L- α-GPC compared with It is few, therefore preparation purity is high, high-quality choline glycerophosphatide product is of great significance.
Enzyme process prepare choline glycerophosphatide refer under certain condition, being capable of hydrolytic phosphatide S using PLBn-lAnd Sn-2Position fat The effect catalysis phosphatidyl choline reaction of fat acid acyl group generates choline glycerophosphatide, and this method has reaction item compared to chemical synthesis The advantages that part is mild, product yield is high, high-quality.Therefore it needs to develop or be transformed to have provided higher active PLB to improve glycerine Phosphocholine production efficiency, to improve production economy benefit.
Invention content
The present invention provides a kind of mutation and its application with phospholipase B activity, using site-directed mutagenesis technique to phosphorus The albumen of lipase B activity is transformed, and improves its efficiency in preparing choline glycerophosphatide.
In order to achieve the above objects and other related objects, the present invention provides a kind of mutant with phospholipase B activity, contains Have and is selected from one of amino acid sequence as follows：
(a)、SEQ ID No：1 to SEQ ID No：Sequence shown in 4；
(b), with sequence shown in (a) at least 90% homogeneity and with the sequence of improved phospholipase B activity；Or
(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 lipase B activity；
Wherein sequence shown in (b) is not SEQ ID No：Sequence shown in 5.
In one embodiment, the mutant with phospholipase B activity contains such as SEQ ID No：Shown in 4 Amino acid sequence.SEQ ID No can also be contained：1, amino acid sequence shown in 2,3.
The invention further relates to the nucleotide coding sequences of phospholipase B mutant as described above, containing as follows Sequence：
(a)、SEQ ID No：Sequence shown in 7-10；
(b), there is at least 90% homogeneity with the sequence described in (a) and polypeptide of the coding with improved phospholipase B activity or The sequence of protein；Or
(c), hybridize under high stringency conditions with the sequence described in (a) and encode the polypeptide with improved phospholipase B activity Or the sequence of protein.
In one embodiment, the nucleotide coding sequence contains such as SEQ ID No：Sequence shown in 9.
The present invention be also related to include a kind of nucleic acid construct or expression vector, it includes described in said program polypeptide or The polynucleotides of protein, the polynucleotides are operably connected to one or more regulating and controlling sequences, and the regulating and controlling sequence refers to Lead the generation of the polypeptide or protein in expression vector.
The present invention is also related to include a kind of recombinant host cell, it includes the polynucleotides in said program, the multinuclear Thuja acid is operably connected to one or more regulating and controlling sequences, and the regulating and controlling sequence instructs the polypeptide or protein to be carried in expression Generation in body.
The present invention provides it is a kind of generate with phospholipase B activity polypeptide or protein method, 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.
The present invention is also related to a kind of composition, and it includes the polypeptides or protein and other enzymes in said program.
The present invention provides a kind of method preparing and detect choline glycerophosphatide, this method includes：
（a）, by the buffer solution containing phosphatidyl choline, pH4.5-9.0, EDTA, Triton X-100 and phospholipase B（Have The wild-type protein or mutant protein of phospholipase B activity）Identical standard reaction system, react 4-30h respectively at 37 DEG C Afterwards, 100 DEG C of water-bath 5min terminate enzyme reaction；
（b）, from（a）In each reaction system take equivalent reaction solution to be added separately to containing glycerophosphocholine phosphodiesterase （GPCP）, Tris-HCl buffer solutions and CaCl2Same reaction system in, react 20min at 37 DEG C；
（c）, from（b）In each reaction system take equivalent reaction solution to be added separately to containing alkaline phosphatase（AP）、glycine- NaOH buffer solutions, MgCl2And ZnCl2Same reaction system in, react 10min at 56 DEG C；
（d）, with BIOMOL Green Reagent phosphate colour reagent boxes detect（c）In the Phos that generates of each reaction system Phosphate content, and the content for the choline glycerophosphatide that the catalysis of above-mentioned phospholipase B generates is the inorganic phosphate being equal in each system Content.
In said program, the related content is explained as follows：
The present invention is to derive fromStreptomyces sp. Sge12The gene of the albumen of the hypothesis with phospholipase B activity of bacterium （Such as SEQ ID No：Shown in 6）For the gene that sets out, gene mutation is carried out, the mutant of activity raising is obtained by directed screening Enzyme.
The present invention's derives fromStreptomyces sp. Sge12The amino of the mutant with phospholipase B activity of bacterium Acid sequence contains following sequence：
(1) SEQ ID No：Amino acid sequence shown in 1：Mutational site is H49K：
MRCASATQRSMHIRGQVHPLPGIDQRVNLACSGAETVNVLSTAAGGQPKLGEAPQTDRLAAVARTARVRLIAL SIGGNDLGFGAIIGDCAYDWYFRRLCWKKQAPVVEQKLPGVRAKVTAVVDDIRATMRAAGYADGDYRLVLQSYPSPI PGGESFRLNQNDSDRMFKDGCPFNDRDADWAAYTLVPRIGDMVEAVAGARGTDYLDLRDALAGHEVCALGPEQVGQT GPDARRHEWFRFLDRVNTQGTLEESMHPNAHGQRAMAVCLGLVGAAAPGRYACTQDWFGDGDPSRMRIRQAV
(2) SEQ ID No：Amino acid sequence shown in 2：Mutational site is G190S：
MRCASATQRSMHIRGQVHPLPGIDQRVNLACSGAETVNVLSTAAGGQPHLGEAPQTDRLAAVARTARVRLIAL SIGGNDLGFGAIIGDCAYDWYFRRLCWKKQAPVVEQKLPGVRAKVTAVVDDIRATMRAAGYADGDYRLVLQSYPSPI PGGESFRLNQNDSDRMFKDGCPFNDRDADWAAYTLVPRISDMVEAVAGARGTDYLDLRDALAGHEVCALGPEQVGQT GPDARRHEWFRFLDRVNTQGTLEESMHPNAHGQRAMAVCLGLVGAAAPGRYACTQDWFGDGDPSRMRIRQAV
(3) SEQ ID No：Amino acid sequence shown in 3：Mutational site is A264V：
MRCASATQRSMHIRGQVHPLPGIDQRVNLACSGAETVNVLSTAAGGQPHLGEAPQTDRLAAVARTARVRLIAL SIGGNDLGFGAIIGDCAYDWYFRRLCWKKQAPVVEQKLPGVRAKVTAVVDDIRATMRAAGYADGDYRLVLQSYPSPI PGGESFRLNQNDSDRMFKDGCPFNDRDADWAAYTLVPRIGDMVEAVAGARGTDYLDLRDALAGHEVCALGPEQVGQT GPDARRHEWFRFLDRVNTQGTLEESMHPNAHGQRAMVVCLGLVGAAAPGRYACTQDWFGDGDPSRMRIRQAV
(4) SEQ ID No：Amino acid sequence shown in 4：Mutational site is G190S+ A264V：
MRCASATQRSMHIRGQVHPLPGIDQRVNLACSGAETVNVLSTAAGGQPHLGEAPQTDRLAAVARTARVRLIAL SIGGNDLGFGAIIGDCAYDWYFRRLCWKKQAPVVEQKLPGVRAKVTAVVDDIRATMRAAGYADGDYRLVLQSYPSPI PGGESFRLNQNDSDRMFKDGCPFNDRDADWAAYTLVPRISDMVEAVAGARGTDYLDLRDALAGHEVCALGPEQVGQT GPDARRHEWFRFLDRVNTQGTLEESMHPNAHGQRAMVVCLGLVGAAAPGRYACTQDWFGDGDPSRMRIRQAV
The coding DNA sequence of above-mentioned phospholipase B mutant includes following DNA sequences：
(1) SEQ ID No：7, it is SEQ ID No：The gene order of hypothesis albumen with phospholipase B activity shown in 6 In the CAC of 145-147bp sport AAA；
(2) SEQ ID No：8, it is SEQ ID No：The gene order of hypothesis albumen with phospholipase B activity shown in 6 In the GGC of 568-570bp sport TCC；
(3) SEQ ID No：9, it is SEQ ID No：The gene order of hypothesis albumen with phospholipase B activity shown in 6 In the GCG of 790-792bp sport GTC；
(4) SEQ ID No：10, it is SEQ ID No：The gene order of hypothesis albumen with phospholipase B activity shown in 6 In the GGC of 568-570bp sport TCC, and the GCG of 790-792bp sports GTC；
Wherein, related definition content is as follows：
Sequence identity：Phase between parameter " sequence identity " two amino acid sequences of description or between two nucleotide sequences Guan Xing.
For the present invention, the degree of sequence identity between two amino acid sequences is by using such as EMBOSS softwares Packet（EMBOSS：The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends in Genetics 16：276-277）（It is preferred that 3.0.0 editions or more highest version）Needle programs in execute Needleman-Wunsch algorithms (Needleman and Wunsch, 1970, J. Mol. Biol. 48：443-453）To measure. The optional parameters used is that gap open penalty (gap open penalty) is 10, gap extension penalty (gap extension Penalty) it is 0.5 and EBLOSUM62（The EMBOSS versions of BLOSUM62）Substitution matrix.Using Needle labeled as " highest is same One property (the output result of longest identity "（It is obtained using-nobrief options）As homogeneity percentage, and calculate It is as follows：
（Same residue × 100）/（Compare the sum of 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 Bi010 Open Software Suite, Rice etc., see above）（It is preferred that 3.0. 0 edition or more highest version）Needle programs in execute Needleman-Wunsch algorithms (Needleman and Wunsch, 1970, it sees above）To measure.The optional parameters used is that gap open penalty is 10, gap extension penalty 0.5 With EDNAFULL (the EMBOSS versions of NCBI NUC4. 4）Substitution matrix.The output of " highest identity " is labeled as using Needle As a result（It is obtained using-nobrief options）As homogeneity percentage, and calculate as follows：
（Same deoxyribonucleotide × 100）/（Compare the sum of notch during length one compares）
High stringency conditions：Term " high stringency conditions " means for length is the probe of at least 100 nucleotide, it then follows mark Quasi- southern blotting technique program, at 42 DEG C in 5X SSPE, 0.3%SDS, 200 micrograms/ml are sheared and are denaturalized toadfish sperm DNA and Prehybridization 12 to 24 hours in 50% formamide.Carrier material finally uses 2X SSC, 0.2%SDS, is washed at 65 DEG C three times, 15 minutes every time.
Coded sequence：Term " coded sequence " means the polynucleotides of directly specified polypeptid acid sequence.Coded sequence Boundary usually determine that the open reading frame is usually with ATG initiation codon or alternative starting by open reading frame Codon such as GTG and TTG start, and are terminated with terminator codon such as TAA, TAG and TGA.Coded sequence can be DNA, CDNA, synthesis or recombination polynucleotides.
cDNA：Term " cDNA " means can be by reverse transcription from the mRNA of the maturation derived from eukaryocyte, own montage DNA molecular prepared by molecule.CDNA lacks the intron sequences being typically found in corresponding gene group DNA.Starting (initial), primary RNA transcript is the precursor of mRNA, passes through a series of step（Including montage）Then processing is made Occur for the mRNA of ripe own montage.
Nucleic acid construct：Term " constructs " means single-stranded or double-stranded nucleic acid molecules, is isolated from and naturally deposits Gene, or it is modified in a manner of being not present in (not otherwise exist) nature originally to contain core Acid section or its be synthesize.When the nucleic acid construct contains the regulating and controlling sequence needed for the coded sequence of the expression present invention When, term nucleic acid construct is synonymous with term " expression cassette ".
Regulating and controlling sequence (control sequence)：Term " regulating and controlling sequence " means the multinuclear glycosides to encoding polypeptide of the present invention Acid expression is required all the components.Each regulating and controlling sequence for the nucleotide sequence of coding said polypeptide can be it is natural or External source or each regulating and controlling sequence is for that can be natural or external source each other.Before these regulating and controlling sequences include but not limited to Lead sequence, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence and transcription terminator.Bottom line regulates and controls sequence Row include the termination signal of promoter and transcription and translation.Regulating and controlling sequence can with for introducing specific restriction sites purpose Connector provide together, the specific restriction sites promote the company of regulating and controlling sequence and the code area for the polynucleotides for encoding polypeptide It connects.
It is operably connected：Term " being operably connected " means such configuration, wherein regulating and controlling sequence is placed in relatively In the appropriate location of the coded sequence of polynucleotides so that regulating and controlling sequence instructs the expression of coded sequence.
Expression：Term " expression " includes any step for being related to polypeptide generation comprising but repaiied after being not limited to transcription, transcription Adorn, fly swiftly translate, posttranslational modification and secretion.
Expression vector：Term " expression vector " means linear or cricoid DNA molecular, and it includes the multinuclears of coding polypeptide Thuja acid, and the polynucleotides are operably connected with the additional nucleotides for being used for its expression are provided.
Host cell：Term " host cell " means that any cell type, the cell type include this hair for using Conversion, transfection, transduction of nucleic acid construct or expression vector of bright polynucleotides etc. are susceptible (susceptible).Term " host cell " covers the offspring of any parental cell, the mutation due to occurring in a replication process and different from parent it is thin Born of the same parents.
The present invention relates to being originated from Streptomyces sp. Sge12The albumen for the hypothesis of bacterium not characterized is pinpointed Mutation, for above-mentioned protein, from its purposes in preparing choline glycerophosphatide or other application is not described.
An embodiment according to the present invention, the inventors discovered that, compare the phospholipase B of the wild albumen mutant Activity significantly improves, and in preparing the method for choline glycerophosphatide using mutant catalysis phosphatidyl choline, conversion effect Rate is remarkably improved.
Production method
WithStreptomyces sp. Sge12The gene of the albumen assumed in bacterial strain is as template（Amino acid sequence such as SEQ ID No：Shown in 5, GeneBank accession number is：WP_081522390.1）, nucleotide primer is designed, is expanded and is obtained by PCR method The mutant of the albumen.Then, the mutant gene obtained is inserted into suitable expression vector, is somebody's turn to do to generate to contain It is assumed that the recombinant vector of the mutant gene of albumen, and the recombinant vector is transformed into suitable host cell.It should The microorganism of conversion carries out shaking flask culture, or small-scale or large scale fermentation (packet is carried out in laboratory or industrial fermentation tank Include continuous, in batches, batch feeding or solid state fermentation) cultivate cell.The culture is to use program as known in the art, Occur in suitable nutrient medium, which includes carbon source and nitrogen source and inorganic salts.Suitable culture medium can be supplied from business Quotient is answered to obtain or can be prepared according to disclosed composition (for example, in catalogue of American type culture collection).If Polypeptide is secreted into the nutrient medium, then polypeptide can be recycled directly from culture medium.If polypeptide is not secreted, It can be recycled from cell pyrolysis liquid.
Polypeptide can be recycled using methods known in the art.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 of recycling.On the one hand, it returns Zymotic fluid of the packet receiving containing the polypeptide.
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.
Specific implementation mode
The technology contents of the present invention are described further with reference to embodiment, but the present invention is not limited solely to these implementations Example cannot limit protection scope of the present invention with following embodiments.And in the following example, the wild type phospholipase B referred to Refer both to described be originated fromStreptomyces sp. Sge12The albumen that bacterium wild type assumes, the present invention's referred to has phosphatidase The mutant of B activity is that amino acid sequence is SEQ ID：Sequence shown in 4.
Embodiment 1：Clone and expression
The gene of coding phosphatidase is cloned from the bacterial strain indicated above shown by routine techniques, or as synthesis Gene is ordered and is inserted into suitable plasmid.
One correct recombination sequence of selection is cloned into expression vector pUC702, conversion to streptomyceteStreptomyces lividans The gene construct is expressed in TK24 host cells.
Embodiment 2：Rite-directed mutagenesis
Protein gene sequence is assumed as template using the wild type of the recombination streptomycete of the expression construct comprising integration, and design is mutated Primer, successively by the 190th glycine mutation be serine, by the 264th alanine mutation be valine, obtained have change Into phospholipase B activity mutant.Then the recombinant bacterium and the correct mutant of sequencing of albumen will be assumed comprising wild type It is cultivated during the conical flask from plating to 250mL is on shaking table respectively, each conical flask includes the 3% of 50ml（w/v） TSB fluid nutrient mediums (thiostrepton for containing 5 μ g/ml).After cultivating 36h at 28 DEG C, 18 800g centrifuge 20min at 4 DEG C Afterwards, it collects supernatant and obtains crude enzyme liquid.
Introducing the rite-directed mutagenesis primer that G190S is mutated is：
Forward primer 5 '-GTCCCGAGGATCTCCGACATGGTCGAGGCGGTGG -3 '
Reverse primer 5 '-CTCGACCATGTCGGAGATCCTCGGGACGAGCGT -3 '
Introducing the rite-directed mutagenesis primer that A264V is mutated is：
Forward primer 5 '-AGCGGGCGATGGTCGTCTGCCTCGGCCTGGTG -3 '
Reverse primer 5 '-GCCGAGGCAGACGACCATCGCCCGCTGCCCGTGG -3 '
Embodiment 3：Enzyme activity detects
（1）Phospholipase B enzyme activity determination method
100 μ l standard detection systems are set, including 50mM Tris-HCl pH=8.0 buffer solutions, 0.5%（w/v）1, 2- dimyristoyl-sn-glycero-3-phosphate, monosodium salt （DMPA）Substrate, 0.5%（w/v） Ttiton X-100,10mM EDTA and 5%（v/v）Phospholipase B, at 50 DEG C after water-bath 5min, 100 DEG C of water-baths 5min terminates reaction.
After reaction, 21 800g centrifuge 5min, according to kit NEFA C Kit (Wako Pure Chemical Industries Ltd) operation instruction detect the free fatty that above-mentioned enzyme reaction system releases using oleic acid as mark product Content.
（3）Enzyme activity is defined as
Using DMPA as substrate, at 50 DEG C, under conditions of pH=8.0, the enzyme amount per minute for generating 1 μm of ol free fatty is defined as One enzyme activity unit, is denoted as U/mL.
（4）The measurement of phospholipase B enzyme activity
The crude enzyme liquid vigor of wild type phospholipase B and phospholipase B mutant is measured, the enzyme activity of mutant before mutation than improving 30%, respectively 25.2U/ml and 32.8U/ml.
Embodiment 4：Choline glycerophosphatide is prepared with wild type phospholipase B and phospholipase B mutant
The reaction system of 100 μ l is set, including 50mM Tris-HCl pH=8.0 buffer solutions, 0.5%（w/v）Phosphatidyl choline, 0.5%（w/v）Ttiton X-100,10mM EDTA and 5%（v/v）Phospholipase B crude enzyme liquid, after reacting 30h at 37 DEG C, 100 DEG C water-bath 5min terminates enzyme reaction.
FromIn take the reaction solution after 50 μ l be added to the GPCP containing 0.17 U, 50mM Tris-HCl pH= In the 100 μ L reaction systems of 9.0 buffer solutions and 15mM CaCl2,20min is reacted at 37 DEG C;
FromIn to take the reaction solution after 10 μ l to be added to the AP containing 2U, pH=9.6 50mM glycine-NaOH slow In the 100ul reaction systems of fliud flushing, 1mM MgCl2 and 0.1 mM ZnCl2,10min is reacted at 56 DEG C；
It is detected with BIOMOL Green Reagent phosphate colour reagent boxesIn after reaction system generate it is inorganic Phosphate content, and the content for the choline glycerophosphatide that the catalysis of above-mentioned phospholipase B generates is the inorganic phosphate being equal in system Content.
The conversion ratio that wherein wild type phospholipase B and phospholipase B mutant enzyme catalysis prepare choline glycerophosphatide is respectively 20% 、52%。
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 and its application with phospholipase B activity
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 299
<212> PRT
<213>Artificial sequence
<400> 1
MRCASATQRS MHIRGQVHPL PGIDQRVNLA CSGAETVNVL STAAGGQPKL GEAPQTDRLA 60
AVARTARVRL IALSIGGNDL GFGAIIGDCA YDWYFRRLCW KKQAPVVEQK LPGVRAKVTA 120
VVDDIRATMR AAGYADGDYR LVLQSYPSPI PGGESFRLNQ NDSDRMFKDG CPFNDRDADW 180
AAYTLVPRIG DMVEAVAGAR GTDYLDLRDA LAGHEVCALG PEQVGQTGPD ARRHEWFRFL 240
DRVNTQGTLE ESMHPNAHGQ RAMAVCLGLV GAAAPGRYAC TQDWFGDGDP SRMRIRQAV 299
<210> 2
<211> 299
<212> PRT
<213>Artificial sequence
<400> 2
MRCASATQRS MHIRGQVHPL PGIDQRVNLA CSGAETVNVL STAAGGQPHL GEAPQTDRLA 60
AVARTARVRL IALSIGGNDL GFGAIIGDCA YDWYFRRLCW KKQAPVVEQK LPGVRAKVTA 120
VVDDIRATMR AAGYADGDYR LVLQSYPSPI PGGESFRLNQ NDSDRMFKDG CPFNDRDADW 180
AAYTLVPRIS DMVEAVAGAR GTDYLDLRDA LAGHEVCALG PEQVGQTGPD ARRHEWFRFL 240
DRVNTQGTLE ESMHPNAHGQ RAMAVCLGLV GAAAPGRYAC TQDWFGDGDP SRMRIRQAV 299
<210> 3
<211> 299
<212> PRT
<213>Artificial sequence
<400> 3
MRCASATQRS MHIRGQVHPL PGIDQRVNLA CSGAETVNVL STAAGGQPHL GEAPQTDRLA 60
AVARTARVRL IALSIGGNDL GFGAIIGDCA YDWYFRRLCW KKQAPVVEQK LPGVRAKVTA 120
VVDDIRATMR AAGYADGDYR LVLQSYPSPI PGGESFRLNQ NDSDRMFKDG CPFNDRDADW 180
AAYTLVPRIG DMVEAVAGAR GTDYLDLRDA LAGHEVCALG PEQVGQTGPD ARRHEWFRFL 240
DRVNTQGTLE ESMHPNAHGQ RAMVVCLGLV GAAAPGRYAC TQDWFGDGDP SRMRIRQAV 299
<210> 4
<211> 299
<212> PRT
<213>Artificial sequence
<400> 4
MRCASATQRS MHIRGQVHPL PGIDQRVNLA CSGAETVNVL STAAGGQPHL GEAPQTDRLA 60
AVARTARVRL IALSIGGNDL GFGAIIGDCA YDWYFRRLCW KKQAPVVEQK LPGVRAKVTA 120
VVDDIRATMR AAGYADGDYR LVLQSYPSPI PGGESFRLNQ NDSDRMFKDG CPFNDRDADW 180
AAYTLVPRIS DMVEAVAGAR GTDYLDLRDA LAGHEVCALG PEQVGQTGPD ARRHEWFRFL 240
DRVNTQGTLE ESMHPNAHGQ RAMVVCLGLV GAAAPGRYAC TQDWFGDGDP SRMRIRQAV 299
<210> 5
<211> 299
<212> PRT
<213> Streptomyces sp. Sge12
<400> 5
MRCASATQRS MHIRGQVHPL PGIDQRVNLA CSGAETVNVL STAAGGQPHL GEAPQTDRLA 60
AVARTARVRL IALSIGGNDL GFGAIIGDCA YDWYFRRLCW KKQAPVVEQK LPGVRAKVTA 120
VVDDIRATMR AAGYADGDYR LVLQSYPSPI PGGESFRLNQ NDSDRMFKDG CPFNDRDADW 180
AAYTLVPRIG DMVEAVAGAR GTDYLDLRDA LAGHEVCALG PEQVGQTGPD ARRHEWFRFL 240
DRVNTQGTLE ESMHPNAHGQ RAMAVCLGLV GAAAPGRYAC TQDWFGDGDP SRMRIRQAV 299
<210> 6
<211> 897
<212> DNA
<213> Streptomyces sp. Sge12
<400> 6
atgcgctgcg cgtccgcgac ccagcggagc atgcacatcc ggggccaggt tcacccgctg 60
cccgggatcg accagcgggt gaacctggcc tgctccggag ccgagacggt caacgtcctg 120
agcacggcgg ccggcgggca gccccacctc ggggaggccc cgcagaccga ccggctggcc 180
gccgtcgcgc ggacggcccg ggtcaggctg atcgcgctgt ccatcggcgg gaacgacctc 240
ggcttcggcg cgatcatcgg cgactgcgcg tacgactggt acttcaggcg gctgtgctgg 300
aagaagcagg cgccggtcgt cgagcagaag ctgcccgggg tgagggccaa ggtcacggcc 360
gtcgtggacg acatccgggc aacgatgcgg gccgccgggt acgcggacgg cgactaccgg 420
ctggtcctgc agtcctaccc gtcaccgatt ccgggcgggg agtccttccg gctgaaccag 480
aacgactcgg accggatgtt caaggacggc tgccccttca acgaccggga cgcggactgg 540
gcggcgtaca cgctcgtccc gaggatcggc gacatggtcg aggcggtggc cggcgcccgc 600
ggcaccgact acctggacct gcgggacgca ctggcggggc acgaggtgtg cgcgctgggc 660
ccggagcagg tggggcagac cggaccggac gcccgccggc acgagtggtt ccgtttcctc 720
gaccgcgtca acacgcaggg cacgctggag gagtccatgc accccaacgc ccacgggcag 780
cgggcgatgg cggtctgcct cggcctggtg ggggcagcgg ctcccggccg gtacgcgtgc 840
acgcaggact ggttcggcga cggggacccg agccggatgc ggatccggca ggccgtg 897
<210> 7
<211> 897
<212> DNA
<213>Artificial sequence
<400> 7
atgcgctgcg cgtccgcgac ccagcggagc atgcacatcc ggggccaggt tcacccgctg 60
cccgggatcg accagcgggt gaacctggcc tgctccggag ccgagacggt caacgtcctg 120
agcacggcgg ccggcgggca gcccaaactc ggggaggccc cgcagaccga ccggctggcc 180
gccgtcgcgc ggacggcccg ggtcaggctg atcgcgctgt ccatcggcgg gaacgacctc 240
ggcttcggcg cgatcatcgg cgactgcgcg tacgactggt acttcaggcg gctgtgctgg 300
aagaagcagg cgccggtcgt cgagcagaag ctgcccgggg tgagggccaa ggtcacggcc 360
gtcgtggacg acatccgggc aacgatgcgg gccgccgggt acgcggacgg cgactaccgg 420
ctggtcctgc agtcctaccc gtcaccgatt ccgggcgggg agtccttccg gctgaaccag 480
aacgactcgg accggatgtt caaggacggc tgccccttca acgaccggga cgcggactgg 540
gcggcgtaca cgctcgtccc gaggatcggc gacatggtcg aggcggtggc cggcgcccgc 600
ggcaccgact acctggacct gcgggacgca ctggcggggc acgaggtgtg cgcgctgggc 660
ccggagcagg tggggcagac cggaccggac gcccgccggc acgagtggtt ccgtttcctc 720
gaccgcgtca acacgcaggg cacgctggag gagtccatgc accccaacgc ccacgggcag 780
cgggcgatgg cggtctgcct cggcctggtg ggggcagcgg ctcccggccg gtacgcgtgc 840
acgcaggact ggttcggcga cggggacccg agccggatgc ggatccggca ggccgtg 897
<210> 8
<211> 897
<212> DNA
<213>Artificial sequence
<400> 8
atgcgctgcg cgtccgcgac ccagcggagc atgcacatcc ggggccaggt tcacccgctg 60
cccgggatcg accagcgggt gaacctggcc tgctccggag ccgagacggt caacgtcctg 120
agcacggcgg ccggcgggca gccccacctc ggggaggccc cgcagaccga ccggctggcc 180
gccgtcgcgc ggacggcccg ggtcaggctg atcgcgctgt ccatcggcgg gaacgacctc 240
ggcttcggcg cgatcatcgg cgactgcgcg tacgactggt acttcaggcg gctgtgctgg 300
aagaagcagg cgccggtcgt cgagcagaag ctgcccgggg tgagggccaa ggtcacggcc 360
gtcgtggacg acatccgggc aacgatgcgg gccgccgggt acgcggacgg cgactaccgg 420
ctggtcctgc agtcctaccc gtcaccgatt ccgggcgggg agtccttccg gctgaaccag 480
aacgactcgg accggatgtt caaggacggc tgccccttca acgaccggga cgcggactgg 540
gcggcgtaca cgctcgtccc gaggatctcc gacatggtcg aggcggtggc cggcgcccgc 600
ggcaccgact acctggacct gcgggacgca ctggcggggc acgaggtgtg cgcgctgggc 660
ccggagcagg tggggcagac cggaccggac gcccgccggc acgagtggtt ccgtttcctc 720
gaccgcgtca acacgcaggg cacgctggag gagtccatgc accccaacgc ccacgggcag 780
cgggcgatgg cggtctgcct cggcctggtg ggggcagcgg ctcccggccg gtacgcgtgc 840
acgcaggact ggttcggcga cggggacccg agccggatgc ggatccggca ggccgtg 897
<210> 9
<211> 897
<212> DNA
<213>Artificial sequence
<400> 9
atgcgctgcg cgtccgcgac ccagcggagc atgcacatcc ggggccaggt tcacccgctg 60
cccgggatcg accagcgggt gaacctggcc tgctccggag ccgagacggt caacgtcctg 120
agcacggcgg ccggcgggca gccccacctc ggggaggccc cgcagaccga ccggctggcc 180
gccgtcgcgc ggacggcccg ggtcaggctg atcgcgctgt ccatcggcgg gaacgacctc 240
ggcttcggcg cgatcatcgg cgactgcgcg tacgactggt acttcaggcg gctgtgctgg 300
aagaagcagg cgccggtcgt cgagcagaag ctgcccgggg tgagggccaa ggtcacggcc 360
gtcgtggacg acatccgggc aacgatgcgg gccgccgggt acgcggacgg cgactaccgg 420
ctggtcctgc agtcctaccc gtcaccgatt ccgggcgggg agtccttccg gctgaaccag 480
aacgactcgg accggatgtt caaggacggc tgccccttca acgaccggga cgcggactgg 540
gcggcgtaca cgctcgtccc gaggatcggc gacatggtcg aggcggtggc cggcgcccgc 600
ggcaccgact acctggacct gcgggacgca ctggcggggc acgaggtgtg cgcgctgggc 660
ccggagcagg tggggcagac cggaccggac gcccgccggc acgagtggtt ccgtttcctc 720
gaccgcgtca acacgcaggg cacgctggag gagtccatgc accccaacgc ccacgggcag 780
cgggcgatgg tcgtctgcct cggcctggtg ggggcagcgg ctcccggccg gtacgcgtgc 840
acgcaggact ggttcggcga cggggacccg agccggatgc ggatccggca ggccgtg 897
<210> 10
<211> 897
<212> DNA
<213>Artificial sequence
<400> 10
atgcgctgcg cgtccgcgac ccagcggagc atgcacatcc ggggccaggt tcacccgctg 60
cccgggatcg accagcgggt gaacctggcc tgctccggag ccgagacggt caacgtcctg 120
agcacggcgg ccggcgggca gccccacctc ggggaggccc cgcagaccga ccggctggcc 180
gccgtcgcgc ggacggcccg ggtcaggctg atcgcgctgt ccatcggcgg gaacgacctc 240
ggcttcggcg cgatcatcgg cgactgcgcg tacgactggt acttcaggcg gctgtgctgg 300
aagaagcagg cgccggtcgt cgagcagaag ctgcccgggg tgagggccaa ggtcacggcc 360
gtcgtggacg acatccgggc aacgatgcgg gccgccgggt acgcggacgg cgactaccgg 420
ctggtcctgc agtcctaccc gtcaccgatt ccgggcgggg agtccttccg gctgaaccag 480
aacgactcgg accggatgtt caaggacggc tgccccttca acgaccggga cgcggactgg 540
gcggcgtaca cgctcgtccc gaggatctcc gacatggtcg aggcggtggc cggcgcccgc 600
ggcaccgact acctggacct gcgggacgca ctggcggggc acgaggtgtg cgcgctgggc 660
ccggagcagg tggggcagac cggaccggac gcccgccggc acgagtggtt ccgtttcctc 720
gaccgcgtca acacgcaggg cacgctggag gagtccatgc accccaacgc ccacgggcag 780
cgggcgatgg tcgtctgcct cggcctggtg ggggcagcgg ctcccggccg gtacgcgtgc 840
acgcaggact ggttcggcga cggggacccg agccggatgc ggatccggca ggccgtg 897
<210> 11
<211> 34
<212> DNA
<213>Artificial sequence
<400> 11
gtcccgagga tctccgacat ggtcgaggcg gtgg 34
<210> 12
<211> 33
<212> DNA
<213>Artificial sequence
<400> 12
ctcgaccatg tcggagatcc tcgggacgag cgt 33
<210> 13
<211> 32
<212> DNA
<213>Artificial sequence
<400> 13
agcgggcgat ggtcgtctgc ctcggcctgg tg 32
<210> 14
<211> 34
<212> DNA
<213>Artificial sequence
<400> 14
gccgaggcag acgaccatcg cccgctgccc gtgg 34

Claims (9)

1. a kind of mutant with phospholipase B activity, it is characterised in that：Containing selected from amino acid sequence as follows it One：
(a)、SEQ ID No：1 to SEQ ID No：Sequence shown in 4；
(b), with sequence shown in (a) at least 90% homogeneity and with the sequence of improved phospholipase B activity；Or
(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 lipase B activity；
Wherein sequence shown in (b) is not SEQ ID No：Sequence shown in 5.
2. the mutant of phospholipase B activity according to claim 1, it is characterised in that：Contain such as SEQ ID No：1、2、3 Or amino acid sequence shown in 4.
3. the mutant of phospholipase B activity according to claim 1 or 2, it is characterised in that：Contain such as SEQ ID No：4 Shown in amino acid sequence.
4. the mutant of phospholipase B activity according to claim 1, it is characterised in that：The phospholipase B mutant Nucleotide coding sequence contains sequence as follows：
(a)、SEQ ID No：Sequence shown in 7-10；
(b), there is at least 90% homogeneity with the sequence described in (a) and polypeptide of the coding with improved phospholipase B activity or The sequence of protein；Or
(c), hybridize under high stringency conditions with the sequence described in (a) and encode the polypeptide with improved phospholipase B activity Or the sequence of protein.
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 in expression vector.
7. a kind of method generating polypeptide or protein with phospholipase B 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. a kind of composition, it includes the polypeptide of any one of claims 1 to 3 or protein and other enzymes.
9. the polypeptide or protein of any one of claims 1 to 3 or the composition of claim 8 are being used to prepare phosphoglycerol courage The method of alkali：
By the buffer solution containing phosphatidyl choline, pH4.5-9.0, EDTA, Triton X-100 and phospholipase B（With phosphatide The wild-type protein or mutant protein of enzyme B activity）Identical standard reaction system, after reacting 4-30h respectively at 37 DEG C, 100 DEG C of water-bath 5min terminate enzyme reaction.
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Cited By (1)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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