CN102286440B - Preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts - Google Patents
Preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts Download PDFInfo
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Abstract
The invention relates to preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts, which belongs to a method for carrying out site directed mutagenesis wild phospholipids enzyme D by recombinant deoxyribonucleic acid (DNA) for improving the activity, connecting the mutated gene with yeast show carriers pPIC9K-Flo and efficiently displaying the mutated gene on the surface of pichia pastoris cells, and relates to a preparation method of the high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts. The method has the advantages that the activity of the wild phospholipids enzyme D is improved and is shown on the surfaces of the pastoris cells, the stability is improved, and the advantages of immobilized enzymes are realized. The method has the technical scheme that wild phospholipids enzyme D genes are separated from microbes, particularly streptomyces aureofuscus, the mutation is carried out on the amino acid residue of Glu69 and Ser285, through the efficient expression on the surfaces of the pichia pastoris cells, the enzyme activity of the high-activity phospholipids enzyme is improved by 11 percent than the wild type phospholipids enzyme D, and the enzyme activity of the recombination strains GS115/pPIC9K-Flo-pldm prepared through high-density fermentation is 120U/(g. stem cells).
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of rite-directed mutagenesis and reorganization of gene, the preparation of especially a kind of high vigor Phospholipase D and cell surface display Phospholipase D yeast whole-cell catalyst.
Background technology
(phosphatidylserine PS) is present in all animals, higher plant and the microbial cell film phosphatidylserine, is one of cell membrane phospholipid composition.PS content in plant-animal and mikrobe is lower, only accounts for the 2-10% of total phospholipids.PS in the human body mainly concentrates on brain, accounts for 15% of big kephalin total amount.PS can only synthesize in human body on a small quantity, and main leaning on absorbed from food.PS regulates the activity of its acceptor, enzyme, ionic channel, messenger molecule etc. through directly acting on film internal protein and embrane-associated protein.PS regulates many metabolic processes (being attached to the enzyme on the cytolemma like activation) except participating in, and also participates in the transmission of nerve information.The appearance of PS has positive prophylactic effect for diseases such as intelligence degeneration that is caused by the age and senile dementias, can improve people's perception and memory.Therefore the synthetic and adjusting of PS receives people's extensive attention.
The main effect of phosphatidylserine (PS) comprises: 1, improve cerebral function; Improve senile dementia: with age growth; PS can reduce with the interior chemical substance of other important brain gradually; Thereby cause memory, cognitive power to weaken, additional PS can increase the mobile of brain bur number, brain cell membrane and promote glucose metabolism in the brain cell, thereby makes brain cell more active; 2, help to repair brain damage, PS is one of staple of brain neuroblastoma, has the activity of various enzymes in nutrition and the activation brain, can delay the minimizing process of neurotransmitter, helps to repair, upgrades the brain damaged cell and removes objectionable impurities; 3, release the pressure, promote recovery, the balance mood of brain fag.
The main stream approach of suitability for industrialized production PS is an organic solvent extractionprocess at present, and have many deficiencies: 1, a large amount of organic solvents of this method consumption have certain hazardness to environment; 2, contain multiple phospholipid composition in the soybean, each phospholipid composition structure is close, similar performance, and PS is less relatively on content, and therefore, the PS purity of being extracted is lower, often contains other phosphatide, like Yelkin TTS, PI, phosphatidylethanolamine etc.; 3, at last also be the problem of most critical be exactly in the raw material content of PS lower; Even if it is that raw material also exists this problem that application contains the soybean of enriching PS; Phospholipid substance in the soybean accounts for the 1.6-2% of soybean gross weight, this wherein PS only account for 0.5-1%, promptly only contain 80-200mgPS in every kilogram of soybean; Add the cost of separation and purification, therefore causing the price of product to continue high.
Therefore the preparation method of PS has become the bottleneck problem of carrying out the PS application and development.Along with people's is to the development trend of raising and China's aging population of health perception, and PS will receive extensive concern, and abroad for the existing development of the Application and Development of PS medicine, China is then at the early-stage.If can improve production technique, utilize enzyme process catalysis to synthesize PS, will be used for treatment and brain diseases associated with low price, high purity, many effects, many advantages such as have no side effect, have wide prospect at field of medicaments.
Because traditional organic extractant solution method exists not enough, production efficiency is extremely low, the begin one's study new synthetic method of PS of people.
Phospholipase D (phospholipaseD; Be called for short PLD) (EC3.1.4.4); The general name that belongs to the class of enzymes of catalysis di(2-ethylhexyl)phosphate fat ester linkage hydrolyzing and base permutoid reaction; Be distributed in from bacterium to high vegeto-animal many biological groups, its major physiological function is to participate in cytolipin metabolism, signal transduction and microbial film formation etc.In recent years, utilize the base permutoid reaction characteristic of Phospholipase D to carry out phospholipid modified and prepare single phosphatide and rare phosphatide makes substantial progress.Therefore, the research work of Phospholipase D more and more receives people's attention.
The research of Phospholipase D has been carried out nearly 50 years, has obtained remarkable effect.But apart from industrial needs, particularly compare with application with other enzyme productions, it almost is blank having very big gap, especially China research work in this respect.Trace it to its cause, mainly contain following some: 1. the enzyme source is narrow and content is atomic in vivo, the quite difficulty of purifying; 2. the poor stability of enzyme and can only in outphasing system, acting on; 3. this enzyme function and mechanism of action is not in vivo got clear as yet fully, and its range of application is wideless.In recent years; Phospholipase D is phospholipid modified and prepare that aspects such as rare phosphatide show great potential and advantage has caused the new research interest of people again; The particularly seed selection of high yield Phospholipase D bacterial classification, the mechanism of action of enzyme and industrial applied research have become current research emphasis and direction.
Free phosphorus ESD poor stability can't be recycled, though the immobilization technology of enzyme can overcome the above problems, traditional process for fixation also can produce some unfavorable factors, and for example, immobilization process possibly cause the activity yield loss of enzyme; In addition because immobilization operation needs use carrier, thereby increased carrier cost and immobilization process cost, and the yeast cell surface display technique to be the immobilization of enzyme provide a kind of biological method based on recombinant gene newly.
The yeast cell surface display technique is a kind of faster eukaryotic protein expression system of development in recent years; Its ultimate principle is external source target protein gene (foreign protein) and specific carrier gene order to be merged the back import yeast cell; Utilize the mechanism of the interior protein transport of yeast cell, make the target protein immobilization be expressed in yeast cell surface to the film surface.At present; Modal yeast surface display expression system comprises: lectin presenting and expressing and the plain presenting and expressing of flocculation; Through the yeast surface display expression technology; Target protein or enzyme immobilization in yeast cell surface, can be improved the stability and the reuse of enzyme, and need not albumen is carried out complicated separation and purification.
Present multiple enzyme comprise lipase, vitamin H ligase enzyme, organophosphor hydrolytic enzyme, Procaine esterase, epimerase, Schardinger dextrins VISOSE saccharase, glycase, cellulase etc. by presenting and expressing successfully at yeast cell surface and biologically active.
Summary of the invention
The object of the present invention is to provide the preparation method of higher high vigor Phospholipase D of a kind of enzyme activity and cell surface display Phospholipase D yeast whole-cell catalyst.
The present invention realizes that the purpose technological line is following:
A kind of high vigor Phospholipase D; The aminoacid sequence of high vigor Phospholipase D is through making the amino acid sites replacement like Glu69, Ser285 in the nucleotide sequence coded wild-type Phospholipase D aminoacid sequence of sequence 7 respectively; Obtain the nucleotide sequence like sequence 8, sequence 8 obtains high vigor Phospholipase D after efficiently expressing at the pichia spp cell surface.
And said wild-type Phospholipase D source is look brown streptomycete AS 4.331.
A kind of cell surface display Phospholipase D Pichia yeast engineering, its building process may further comprise the steps:
(1) the mature peptide gene of rite-directed mutagenesis wild-type Phospholipase D obtains high vigor Phospholipase D gene;
(2) high vigor Phospholipase D gene order is linked to each other with carrier, obtain carrying the recombinant vectors of high vigor Phospholipase D gene;
(3) recombinant vectors is transformed in the host strain pichia spp, obtains the Pichia yeast engineering of cell surface display Phospholipase D.
And described recombinant vectors is pichia spp display carrier pPIC9K-Flo.
And described host cell is pichia spp GS115.
The pichia spp whole-cell catalyst of the high vigor Phospholipase D of a kind of cell surface display is characterized in that: the Yeast engineering bacteria that uses is cell surface display Phospholipase D Yeast engineering bacteria.
And the preparation process may further comprise the steps:
The Yeast engineering bacteria that (1) will be incubated on the YPD Agr solid plate is seeded in the YPD liquid nutrient medium; 30 ℃, 250r/min are cultivated 24h, are transferred in the fresh BMGY substratum with the inoculum size of 1% (m/m), and 30 ℃, 250r/min are cultivated 24h; Centrifugal 10min obtains thalline at 4 ℃, 8000r/min then; Change in the BMMY substratum, 30 ℃, 250r/min are cultivated 5d, and every to add final concentration at a distance from 24h be that the 0.5%V/V methanol induction produces enzyme.
(2) collect fermented liquid then, centrifugal, get bacterial sediment, wash 2 times with distilled water, use a small amount of distilled water resuspended at last, with protective material, obtain whole-cell catalyst dry powder through vacuum lyophilization.
Advantage of the present invention and beneficial effect are:
1, the present invention uses overlapping pcr; The wild-type Phospholipase D is carried out rite-directed mutagenesis (Gly69Glu, Ser285Ala), make up yeast surface display carrier pPIC9K-Flo-pldm, transform pichia spp; Make Phospholipase D be illustrated in yeast surface; High vigor Phospholipase D has improved 11% than wild-type Phospholipase D vigor under 37 ℃.
2, the present invention adopts the yeast display systems; Phospholipase D is showed in yeast surface, and yeast cell both as the producer of enzyme, can serve as the carrier in the immobilized enzyme again; The purifying of having removed enzyme from waits operation with fixing; Simplified production link, reduced production cost, made the yeast display technique become the developing direction that has potentiality that enzyme process catalysis Yelkin TTS generates phosphatidylserine.
3, Phospholipase D catalyzed reaction of the present invention is carried out in biphasic system, and for resolvase, organic solvent usually reduces its vigor in nonaqueous phase catalysis, shows that enzyme has effectively improved above-mentioned shortcoming.
4, the present invention has improved the vigor of Phospholipase D; And it is illustrated in the pichia spp cell surface; Improve its stability; Advantage with immobilized enzyme improves 11% through detecting the work of high vigor Phospholipase D enzyme than wild-type, and the whole-cell catalyst enzyme of recombinant bacterial strain GS115/pPIC9K-Flo-pldm high density fermentation preparation is lived and is 120U/ (g stem cell).
Description of drawings
Fig. 1 is Phospholipase D mature peptide gene PCR amplified production electrophorogram of the present invention (wherein 1 for DNA Marker, 2 for be the Phospholipase D mature peptide gene that template arrives through pcr amplification with look brown streptomyces gene group DNA);
Fig. 2 is the building process of pPIC9K-Flo-pldm plasmid of the present invention;
Embodiment
Below in conjunction with embodiment technology contents of the present invention is further specified; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
One, the acquisition of wild-type Phospholipase D mature peptide gene
1, wild-type Phospholipase D mature peptide gene is purchased the institute of microbiology in the Chinese Academy of Sciences from look brown streptomycete (AS 4.331), extracts its genomic dna.
Wherein the extraction step of look brown streptomyces gene group DNA is following:
The bacterium liquid that (1) will be cultured to logarithmic phase is got 1mL, and the centrifugal 1min of 12000r/min collects thalline.
(2) thalline is suspended in 90 μ L ddH
2Among the O, add 50mg/mL N,O-Diacetylmuramidase 10 μ L, fully 37 ℃ of water bath heat preservation 20min behind the mixing.
(3) add 400 μ L lysate mixings to producing significant foam, add 5mol/L NaCl 200 μ L, put upside down mixing gently, the centrifugal 10min of 1200r/min.
(4) supernatant is transferred in another Ep pipe, adds 1/2 volume phenol and 1/2 volume chloroform, gentle mixing.The centrifugal 3min of 12000r/min.
(5) get supernatant, add phenol, the chloroform extracting repeatedly of 1/2 volume, centrifugal, suct clearly, repeat this step till two-phase interface can't see white mass.
(6) add isopyknic chloroform and carry out extracting, centrifugal, suct clearly.
(7) with the absolute ethyl alcohol-70 ℃ about 30min of deposit D NA of two volumes, the centrifugal 8min of 12000r/min, 200 μ L70% washing with alcohol twice, the centrifugal 5min of 12000r/min, room temperature is dried.
(8) DNA is dissolved in 20 μ LddH
2-20 ℃ of short-terms are preserved among the O.
2, according to reporting Phospholipase D mature peptide gene, analyze its conserved sequence, the amplimer that designs Phospholipase D mature peptide gene of the present invention is following:
Upper reaches P1:CCG
ACGCGTGCCGACCAGGCGCCCGCCTTCCT (underscore partly is the MluI restriction enzyme site)
Downstream P2:CCG
GAATTCCtacttatcgtcgtcatccttgtaatcCACGGGGTCGTAGGTGCGC (underscore partly is the EcoRI restriction enzyme site, and small letter is the flag label)
Amplification template is look brown streptomyces gene group DNA, and the reaction conditions of its amplification is: 95 ℃ of 5min; 94 ℃ of 1min, 60 ℃ of 1min40s, 72 ℃ of 1min, 30 circulations; 72 ℃ prolong 10min;
Amplification system 50 μ L:ddH
2O 7.5 μ L, 2 * buffer, 25 μ L, dNTPs 5mmol/L, upstream primer 10 μ mol/L 5 μ L, downstream primer 10 μ mol/L 5 μ L, dna profiling 2 μ L, LA TaqDNA polysaccharase 0.5 μ L;
Pcr amplification product obtains the band (Fig. 1) about 1500bp through 0.8% agarose gel electrophoresis, reclaims test kit with miniprep dna and reclaims the PCR product, obtains wild-type Phospholipase D mature peptide gene pld of the present invention, sees sequence 7.
Two, high vigor Phospholipase D gene obtains.
(1) wild-type Phospholipase D gene connects into the T carrier.
The goal gene that pcr amplification is obtained carries out purifying; It is connected into the T carrier; Utilize electrotransformation that this recombinant plasmid is changed in the e. coli jm109, EcoRI, MluI double digestion and PCR checking result show that wild-type Phospholipase D gene successfully is cloned on the T carrier.
Its order-checking can be known the Phospholipase D gene order that increases, see sequence 7.
(2) rite-directed mutagenesis
Carry out rite-directed mutagenesis based on overlapping pcr, make up high vigor Phospholipase D gene.Gly69 → Glu, Ser285 → Ala design primer is following:
Upper reaches CCG
ACGCGTGCCGACCAGGCGCCCGCCTTCCT (underscore partly is the MluI restriction enzyme site)
Downstream CCG
GAATTCCtacttatcgtcgtcatccttgtaatcCACGGGGTCGTAGGTGCGC (underscore partly is the EcoRI restriction enzyme site, and small letter is the flag label)
Overlapping primer P3:5 '-cacgcgccgagagcgaccacacc-3 '
Overlapping primer P4:5 '-ggtgtggtcgctctcggcgcgtg-3 '
Overlapping primer P5:5 '-caggccgccacggccagcggt-3 '
Overlapping primer P6:5 '-accgctggccgtggcggcctg-3 '
Introduce MluI and EcoRI restriction enzyme site respectively at upstream primer and downstream primer 5 ' end.Overlapping primer P3 and overlapping primer P4 are complementary, and overlapping primer P5 and overlapping primer P6 are complementary.Comprised sudden change among overlapping primer P3 and the P4 to 69 amino acids residues.Then comprised sudden change among overlapping primer P5 and the P6 to 285 amino acids residues.
Gly69→Glu
With primer P1, the P4 upper reaches fragment pld1 that increases, P3, the P2 downstream fragment pld2 that increases.
The PCR reaction system comprises: 2 * buffer, 25 μ L, and dNTP 2 μ L, upstream primer P1, P3/ downstream primer P2, each 5 μ L of P4, recombinant plasmid pUC-T-pld 100ng, LA Taq archaeal dna polymerase 0.5 μ L, aseptic deionized water is supplemented to 50 μ L.
The pcr amplification condition is: 94 ℃ of preparatory sex change 5min, totally 1 circulation; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min40s, 72 ℃ are extended 1min, totally 30 circulations; 72 ℃ are extended 10min, totally 1 circulation.
The PCR product is cut glue and is reclaimed, suitably dilution.With pld1, pld2 is primer, carries out PCR.The PCR reaction system is: 2 * buffer25 μ L, and dNTP 2 μ L, pld1, each 1 μ L of pld2, LA Taq archaeal dna polymerase 0.5 μ L, aseptic deionized water is supplemented to 48 μ L.The pcr amplification condition is: 94 ℃ of preparatory sex change 5min, 1 circulation; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min40s, 72 ℃ are extended 1min, 5 circulations; Add primer P1, each 1 μ L of P2, carry out PCR, the pcr amplification condition is: 94 ℃ of preparatory sex change 5min, 1 circulation; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min40s, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 10min, totally 1 circulation.
The PCR reaction solution is carried out 1% agarose gel electrophoresis, cut glue and reclaim target DNA fragment.Obtain the Phospholipase D two mutants of Gly69 → Glu.
Ser285→Ala
Use Phospholipase D mutator gene Gly69 → Glu to be template, primer P1, the P6 upper reaches fragment pld3 that increases, P5, the P2 downstream fragment pld4 that increases.
The PCR reaction system comprises: 2 * buffer, 25 μ L, and dNTP 2 μ L, upstream primer P1, P3/ downstream primer P2, each 5 μ L of P4, Phospholipase D mutator gene Gly69 → Glu 2 μ L, LA Taq archaeal dna polymerase 0.5 μ L, aseptic deionized water is supplemented to 50 μ L.
The pcr amplification condition is: 94 ℃ of preparatory sex change 5min, totally 1 circulation; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min40s, 72 ℃ are extended 1min, totally 30 circulations; 72 ℃ are extended 10min, totally 1 circulation.
The PCR product is cut glue and is reclaimed, suitably dilution.With pld3, pld4 is primer, carries out PCR.The PCR reaction system is: 2 * buffer25 μ L, and dNTP 2 μ L, pld3, each 1 μ L of pld4, LA Taq archaeal dna polymerase 0.5 μ L, aseptic deionized water is supplemented to 48 μ L.The pcr amplification condition is: 94 ℃ of preparatory sex change 5min, 1 circulation; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min40s, 72 ℃ are extended 1min, 5 circulations; Add primer P1, each 1 μ L of P2, carry out PCR, the pcr amplification condition is: 94 ℃ of preparatory sex change 5min, 1 circulation; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min40s, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 10min, totally 1 circulation.
The PCR reaction solution is carried out 1% agarose gel electrophoresis, cut glue and reclaim target DNA fragment.Obtain the high vigor Phospholipase D gene pldm of Gly69 → Glu, Ser285 → Ala, see sequence 8.
Three, the structure of Phospholipase D genetic engineering bacterium
1, the structure of recombinant plasmid pPIC9K-Flo-pldm and evaluation
Overlapping PCR purified product and carrier pPIC9K-Flo behind MluI and EcoRI double digestion, are reclaimed test kit with miniprep dna respectively and reclaim enzyme and cut product, and the Solution I that Application of DNA connects test kit connects 12h under 16 ℃ condition; High vigor Phospholipase D gene orientation is connected to carrier pPIC9K-Flo; Product applied chemistry conversion method be will connect and screening and culturing in containing the LA substratum of Amp, picking positive transformant will be transformed in the E.coliDH5 α competence; Cultivate back upgrading grain; Identify order-checking, called after pPIC9K-Flo-pldm with MluI and EcoRI double digestion.
The competent preparation method of intestinal bacteriaization commentaries on classics wherein:
(1) single bacterium colony of picking from the intestinal bacteria flat board is inoculated in the 2mL LB substratum test tube 37 ℃ of 180r/min shaking table overnight cultures;
(2) get 500 μ L bacterium liquid and be transferred in the 250mL Erlenmeyer flask that contains 50mL LB substratum, 37 ℃ of 180r/min shaking tables are cultivated 2～3h.
(3) bacterium liquid is transferred in the 50mL centrifuge tube, placed 10min on ice;
(4) 4 ℃, the centrifugal 10min of 4000r/min reclaim somatic cells, pour out nutrient solution, will manage and be inverted 1min, so that liquid nutrient medium flows to end;
(5) with ice-cold 0.1mol/LCaCl
2The solution 10mL deposition that suspends is placed on immediately and is incubated 30min on ice;
(6) 4 ℃, the centrifugal 10min of 4000r/min reclaim somatic cells;
(7) with ice-cold 0.1mol/LCaCl
2With 15% glycerine mixed solution 2mL re-suspended cell;
(8) packing cell, every part 50 μ L promptly obtains competent cell.
Wherein the competent concrete steps of recombinant plasmid pPIC9K-Flo-pldm chemical conversion E.coli DH5 α are:
(1) adds 10 μ L DNAs and connect product in 100 μ L competence, fully mixing;
(2) place 30min on ice, 42 ℃ of water-bath 90s;
(3) place 1～2min on ice;
(4) competent cell is added in the 800 μ LLB substratum, 37 ℃ of shaking tables, 1h is cultivated in the 180r/min recovery;
(5) after recovery is cultivated, get the centrifugal 2min of bacterium liquid 8000r/min, sop up 1/3 supernatant after, thalline is resuspended, coat the Amp resistant panel, be inverted for 37 ℃ and cultivate 12～16;
2, the structure of pichia spp GS115 genetic engineering bacterium
The recombinant plasmid that order-checking is correct is after the SalI linearizing, and the LiCl method transforms pichia spp GS115, MD plate screening recon, and the picking positive transformant carries out abduction delivering.
Wherein the competent preparation method of pichia spp GS115 is:
(1) streak culture on the YPD flat board, picking list bacterium colony is inoculated in the 50mLYPD liquid nutrient medium, and 30 ℃, the 250r/min shaking culture is to OD
600=0.8～1.2.
(2) collect somatic cells, with the aseptic water washing of 30mL 3 times, 4 ℃, the centrifugal 10min of 5000r/min.
(3) behind the Ex-all sterilized water with the resuspended gently somatic cells of the LiCl of lmL 0.1mol/L.
(4) in ultra-clean work, the somatic cells suspension liquid is transferred in the aseptic centrifuge tube of 1.5mL.
(5) the centrifugal 1min of room temperature 12000r/min, the deposition somatic cells is also with aseptic rifle head Ex-all LiCl.
(6) the resuspended somatic cells of LiCl of adding 400 μ L 0.1mol/L.
(7) the cell suspension branch with 50 μ L installs in the aseptic centrifuge tube of 1.5mL.
Wherein the competent concrete steps of recombinant plasmid pPIC9K-Flo-pldm chemical conversion GS115 are:
(1) boils single stranded DNA 5min, place cooled on ice fast.
(2) get the centrifugal 10min Ex-all of freshly prepd competent cell 12000r/min LiCl.
(3) sample with each conversion is sequentially added into following reagent: the PEG of 240 μ L 40%, 36 μ L 1M LiCl, 10 μ LDNA.
(4) violent vortex cell precipitation is to its complete mixing (approximately needing about 3min).
(5) static placement 30min in 30 ℃ water-bath.
(6) thermal shock 20～25min in 42 ℃ water-bath.
(7) 30 ℃, the centrifugal 5min of 6000r/min, Ex-all supernatant solution.
(8) with the resuspended somatic cells deposition of the sterilized water of 0.5mL.
(9) the re-suspended cell solution of even coating 100～300 μ L on the MD flat board is inverted cultivation 48～96h at 30 ℃.
Wherein the concrete grammar of abduction delivering is:
Recombinant yeast pichia pastoris pPIC9K-Flo-pldmGS115 is inoculated in 20mL to be contained in the BMGY substratum of 0.5% glycerine; 28 ℃; 250r/min shaking culture 24h; Every then methyl alcohol that in substratum, adds final concentration 0.5% (V/V) at a distance from 24h continues at 28 ℃ shaking culture 120h under the 250r/min condition.
Four, the mensuration of the preparation of whole-cell catalyst and hydrolysis vigor thereof
1, the preparation of whole-cell catalyst
The pichia spp recombinant bacterial strain that (1) will be incubated on the YPD Agr solid plate is seeded in the YPD liquid nutrient medium; 30 ℃, 250r/min are cultivated 24h, and the inoculum size with 1% is transferred in the fresh BMGY substratum, and 30 ℃, 250r/min are cultivated 24h; Centrifugal 10min obtains thalline at 4 ℃, 8000r/min then; Change in the BMMY substratum, 30 ℃, 250r/min are cultivated 5d, and every to add final concentration at a distance from 24h be that 0.5% (V/V) methanol induction produces enzyme.
(2) collect fermented liquid then, centrifugal, get bacterial sediment, wash 2 times with distilled water, use a small amount of distilled water resuspended at last, with protective material, obtain whole-cell catalyst dry powder through vacuum lyophilization.
2, the mensuration of whole-cell catalyst hydrolysis vigor
Adopt enzyme to join colourimetry and carry out the activity detection: Phospholipase D catalytic hydrolysis L-α-Yelkin TTS generates choline; Choline generates hydrogen peroxide under the effect of E.C. 1.1.99.1; Hydrogen peroxide generates the quinonimine substance that show color with amino antipyrine of 4-and phenol under the effect of peroxidase, under A=500nm, have light absorption value.Reaction system: the mixed system of ethanolic soln (dense eventually is 1%) of deionized water, 0.272mL17.9% that L-α-Yelkin TTS of 220mg is dissolved in the SDS solution that contains 3mL 50mM, 6mL 1M NaOAc damping fluid (pH=8.0), 39mL is as the substrate lysate.The amino antipyrine of 39mg 4-, 80mg phenol and 8mg px are dissolved in 100mM Tris HCl (pH=8) damping fluid of 5.5mL as the choline developer.Get the 500mM CaCl of 2.4mL substrate solution, 0.3mL
2The deionized water mixing of solution, 0.2mL also places 37 ℃ of water-baths.The enzyme liquid that adds 0.1mL then; Mix, in 37 ℃ of reaction 10min, boiling water bath termination reaction; To be cooled to room temperature adding 0.05mL 2M Tris-HCl (pH=9) damping fluid; After mixing was also centrifugal, supernatant was got filtrating 2mL and 0.1mL choline developer, 0.1mL E.C. 1.1.99.1 solution room temperature reaction 2.5h with 0.45 μ m filtering with microporous membrane.In reaction mixture, add the 2.0mL deionized water, centrifugal obtaining clarified bright pink solution, at last in A
500nmDetect light absorption value.Enzyme is lived definition: pH=8.0, T=37 ℃ the time, and catalytic hydrolysis L-α-Yelkin TTS discharges the needed enzyme amount of choline of 1.0 μ mol in the Phospholipase D 1min.
The whole-cell catalyst enzyme that records the high vigor Phospholipase D of surface display is lived to 120U/ (g stem cell), has improved 11% than the wild-type Phospholipase D.
SEQUENCE?LISTING
< 110>University Of Science and Technology Of Tianjin
< 120>preparation of high vigor Phospholipase D and cell surface display Phospholipase D yeast whole-cell catalyst
<130>2011-07-14
<160>8
<170>PatentIn?version?3.3
<210>1
<211>32
<212>DNA
< 213>P1 upstream primer sequence
<400>1
ccgac?gcgtg?ccgac?caggc?gcccg?ccttc?ct 32
<210>2
<211>52
<212>DNA
< 213>P2 downstream primer sequence
<400>2
ccgga?attcc?tactt?atcgt?cgtca?tccttgtaat?ccacg?gggtc?gtagg?tgcgc 55
<210>3
<211>23
<212>DNA
< 213>primer sequence P3
<400>3
cacgc?gccga?gagcg?accac?acc 23
<210>4
<211>23
<212>DNA
< 213>primer sequence P4
<400>4
ggtgt?ggtcg?ctctc?ggcgc?gtg 23
<210>5
<211>21
<212>DNA
< 213>primer sequence P5
<400>5
caggc?cgcca?cggcc?agcgg?t 21
<210>6
<211>21
<212>DNA
< 213>primer sequence P6
<400>6
accgc?tggcc?gtggc?ggcct?g 21
<210>7
<211>1533
<212>DNA
< 213>wild-type Phospholipase D mature peptide gene
<400>7
gccgaccagg?cgcccgcctt?cctgcacggc?gtcgcctccg?gtgacccgct?gcccgacggc 60
gtcctgctgt?ggacccgggt?gaccccggtg?cccgaggcga?tacccggctc?cggagtgggc 120
ccggacaccg?aggtcggctg?ggtcgtcgcc?cgcgacaagg?cgttcaccga?cgtcgtcgcc 180
aagggctcga?ccaccgcacg?cgccgggagc?gaccacaccg?tcaaggccga?catccgcggc 240
ctggccccgg?ccaccgacta?ctggttccgc?ttcacggccg?gcggcacgga?ctccccggtg 300
gcgcgcaccc?gcaccgcgcc?ggcggcggac?gcggcggtgt?cctccctgcg?cttcggcgtg 360
gtgtcctgcg?ccaactggga?ggcgggctac?ttcgccgcct?accgccacct?cgcggcccgc 420
aacgacctgg?acgcctggct?gcacctcggc?gactacatct?acgagtacaa?gtccggcgag 480
tacgcggccc?ggggcaccgt?cgtgcggccg?cacgcgccgg?ccaacgagat?cctcaccctc 540
gccgactacc?gcacccggca?cgccaagtac?aagaccgacc?ccgacctgca?ggccctgcac 600
ctgaaggcgc?cggtcatcgc?gatctgggac?gaccacgagt?tcgccgacaa?cgcctggtcc 660
ggcggcgcgg?tgaaccacac?cgagggcgcc?gagggcacct?ggtcggcccg?tcaggccgcc 720
gccaagcagg?cctacttcga?gtggatgccg?gtgcgccccg?ccatcgccgg?caccacctac 780
cggcggctgc?gcttcggcaa?gctcgccgac?ctctccctgc?tggacctgcg?ctccttccgc 840
tcccagcagg?cctccacggc?cagcggttcg?gtggacgacc?cggaccgtac?gctcaccggc 900
cgcgcgcagc?tcgactggct?caaggccggc?ctgaaggcct?ccgacaccag?gtggcggctg 960
gtcggcaact?ccgtgatgat?ctcgccgttc?gccgtcggct?cactctccgc?cgacctgctc 1020
aagccgctcg?ccaagctgct?gggcctgccg?caggagggca?tcgccgtcaa?cacgacccag 1080
tgggacggct?acaccgacga?ccggcgcgaa?ctcctcgccc?acctgcgctc?gaacgccatc 1140
ggcaacaccg?tcttcctgac?cggtgacatc?cacatggcgt?gggccaacga?cgtgccggtg 1200
gacgcgggca?cctatccgct?gtcggcgtcc?gcggccaccg?agttcgtggt?cacgtcggtc 1260
acctccgaca?acctcgacga?catcgtgaag?gtgcccgagg?gcaccgtctc?ggccgtcgcc 1320
tcgccggtca?tcaaggccgc?caaccggcac?gtccactggg?tggacaccga?ccggcacggc 1380
tacggcgtgc?tggacatcac?cgccgaccgc?gcgcagatgg?actactacgt?gctgtccgac 1440
cgcaccgacg?cgaacgcgac?ctcggcgtgg?gtgcggtcgt?accgcacgcg?cagcggcacg 1500
cagcgggtag?agcgcaccta?cgaccccgtg?tag 1533
<210>8
<211>1533
<212>DNA
< 213>high vigor Phospholipase D gene
<400>8
gccgaccagg?cgcccgcctt?cctgcacggc?gtcgcctccg?gtgacccgct?gcccgacggc 60
gtcctgctgt?ggacccgggt?gaccccggtg?cccgaggcga?tacccggctc?cggagtgggc 120
ccggacaccg?aggtcggctg?ggtcgtcgcc?cgcgacaagg?cgttcaccga?cgtcgtcgcc 180
aagggctcga?ccaccgcacg?cgccgagagc?gaccacaccg?tcaaggccga?catccgcggc 240
ctggccccgg?ccaccgacta?ctggttccgc?ttcacggccg?gcggcacgga?ctccccggtg 300
gcgcgcaccc?gcaccgcgcc?ggcggcggac?gcggcggtgt?cctccctgcg?cttcggcgtg 360
gtgtcctgcg?ccaactggga?ggcgggctac?ttcgccgcct?accgccacct?cgcggcccgc 420
aacgacctgg?acgcctggct?gcacctcggc?gactacatct?acgagtacaa?gtccggcgag 480
tacgcggccc?ggggcaccgt?cgtgcggccg?cacgcgccgg?ccaacgagat?cctcaccctc 540
gccgactacc?gcacccggca?cgccaagtac?aagaccgacc?ccgacctgca?ggccctgcac 600
ctgaaggcgc?cggtcatcgc?gatctgggac?gaccacgagt?tcgccgacaa?cgcctggtcc 660
ggcggcgcgg?tgaaccacac?cgagggcgcc?gagggcacct?ggtcggcccg?tcaggccgcc 720
gccaagcagg?cctacttcga?gtggatgccg?gtgcgccccg?ccatcgccgg?caccacctac 780
cggcggctgc?gcttcggcaa?gctcgccgac?ctctccctgc?tggacctgcg?ctccttccgc 840
tcccagcagg?ccgccacggc?cagcggttcg?gtggacgacc?cggaccgtac?gctcaccggc 900
cgcgcgcagc?tcgactggct?caaggccggc?ctgaaggcct?ccgacaccag?gtggcggctg 960
gtcggcaact?ccgtgatgat?ctcgccgttc?gccgtcggct?cactctccgc?cgacctgctc 1020
aagccgctcg?ccaagctgct?gggcctgccg?caggagggca?tcgccgtcaa?cacgacccag 1080
tgggacggct?acaccgacga?ccggcgcgaa?ctcctcgccc?acctgcgctc?gaacgccatc 1140
ggcaacaccg?tcttcctgac?cggtgacatc?cacatggcgt?gggccaacga?cgtgccggtg 1200
gacgcgggca?cctatccgct?gtcggcgtcc?gcggccaccg?agttcgtggt?cacgtcggtc 1260
acctccgaca?acctcgacga?catcgtgaag?gtgcccgagg?gcaccgtctc?ggccgtcgcc 1320
tcgccggtca?tcaaggccgc?caaccggcac?gtccactggg?tggacaccga?ccggcacggc 1380
tacggcgtgc?tggacatcac?cgccgaccgc?gcgcagatgg?actactacgt?gctgtccgac 1440
cgcaccgacg?cgaacgcgac?ctcggcgtgg?gtgcggtcgt?accgcacgcg?cagcggcacg 1500
cagcgggtag?agcgcaccta?cgaccccgtg?tag 1533
Claims (3)
1. the Pichia yeast engineering of a cell surface display Phospholipase D, it is characterized in that: its building process may further comprise the steps:
⑴ the wild-type Phospholipase D mature peptide gene shown in the rite-directed mutagenesis sequence 7 obtains the Phospholipase D gene shown in the sequence 8;
⑵ link to each other the Phospholipase D gene shown in the sequence 8 with pichia spp display carrier pPIC9K-Flo, obtain recombinant vectors;
⑶ the recombinant vectors that obtain step (2) is transformed among the host strain pichia spp GS115, obtains the Pichia yeast engineering of cell surface display Phospholipase D.
2. the pichia spp whole-cell catalyst of a cell surface display Phospholipase D, it is characterized in that: the Yeast engineering bacteria that uses is the Pichia yeast engineering of the described cell surface display Phospholipase D of claim 1.
3. the pichia spp whole-cell catalyst of cell surface display Phospholipase D according to claim 2 is characterized in that: the preparation process may further comprise the steps:
⑴ the Yeast engineering bacteria that will be incubated on the YPD Agr solid plate is seeded in the YPD liquid nutrient medium; 30 ℃, 250r/min are cultivated 24h, and the inoculum size with 1% is transferred in the fresh BMGY substratum, and 30 ℃, 250r/min are cultivated 24h; Centrifugal 10min obtains thalline at 4 ℃, 8000r/min then; Change in the BMMY substratum, 30 ℃, 250r/min are cultivated 5d, and every to add final concentration at a distance from 24h be that 0.5% (V/V) methanol induction produces enzyme;
⑵ collect fermented liquid then, centrifugal, gets bacterial sediment, washes 2 times with distilled water, uses a small amount of distilled water resuspended at last, with protective material, obtains whole-cell catalyst dry powder through vacuum lyophilization.
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| CN104017786B (en) * | 2014-06-20 | 2016-06-15 | 天津科技大学 | A kind of phospholipase A2Mutant and preparation method thereof |
| CN104004797B (en) * | 2014-06-20 | 2016-03-16 | 天津科技大学 | Sn-2 position is the preparation method of the phosphatidylserine of docosahexenoic acid |
| CN110129299B (en) * | 2016-06-02 | 2021-08-03 | 天津科技大学 | Phospholipase D and application thereof |
| WO2020133314A1 (en) * | 2018-12-29 | 2020-07-02 | 邦泰生物工程(深圳)有限公司 | Phospholipase d mutant, use thereof and method for preparing phosphatidylserine by means of same |
| CN112575023B (en) * | 2020-12-31 | 2023-08-22 | 河南省商业科学研究所有限责任公司 | Method for efficiently expressing phospholipase D in streptomyces and recombinant streptomyces |
| CN116790553B (en) * | 2023-08-25 | 2023-11-10 | 中国海洋大学 | Phospholipase D-PKV-S109L and application thereof |
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