CN101319237A - Method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase - Google Patents
Method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase Download PDFInfo
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- CN101319237A CN101319237A CNA2008100538127A CN200810053812A CN101319237A CN 101319237 A CN101319237 A CN 101319237A CN A2008100538127 A CNA2008100538127 A CN A2008100538127A CN 200810053812 A CN200810053812 A CN 200810053812A CN 101319237 A CN101319237 A CN 101319237A
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Abstract
The invention relates to a method for catalytic synthesis of phosphatidylserine by phosphatidylserine synthetase. The technical proposal of the method comprises the following steps that: (1) a phosphatidylserine synthetase liquid is added into an acetic acid buffer liquid containing serine, CaCl2 and Triton-X100, then the mixture is mixed with a chloroform solution containing lecithin according to the equal volume to react for 3 to 5 hours at a temperature of 30 DEG C; (2) double liquid phases are naturally layered after the reaction is finished, and an organic phase is extracted for standby; and (3) after the organic phase is naturally volatilized, the remainder is washed by chloroform to produce the phosphatidylserine. The method utilizes the high specificity of the phosphatidylserine synthetase to catalyze the lecithin and the serine to prepare the phosphatidylserine, which effectively solves the problem that the prior extraction method and chemical synthesis method fail to obtain high-purity phosphatidylserine, has steady and reliable process and high efficiency, is suitable for industrialized production, and has large application potential in medical and health food fields.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of method of catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase.
Background technology
Phospholipase D, i.e. phosphatidylcholine phosphatide lytic enzyme (EC3.1.4.4) belongs to the general name of the class of enzymes of catalysis di(2-ethylhexyl)phosphate fat ester linkage hydrolyzing and base permutoid reaction, and its major physiological function is to participate in cytolipin metabolism, signal transduction and microbial film formation etc.
(phosphatidylserine synthase PSS) is one of Phospholipase D family member to phosphatidylserine synthetase, and its nucleotide sequence all has discovery at present in bacterium, yeast and mammalian cell shown in SEQ ID NO.1.Wherein, be derived from colibacillary phosphatidylserine synthetase and have this family's obvious sequence feature, promptly contain two H (x) K (x) that occur at interval
4The D conserved sequence, it can catalysis synthesize the required most of phospholipid substance of intestinal bacteria self, and is different from the phospholipid substance synthetic enzyme of other Gram-negative bacteria, closely is not connected in membrane structure.Its further Position Research is found that this enzyme is a kind of outer membrane protein, and mainly electron transport chain and the film surface by lipid substrate and slightly acidic phospholipid material interacts.
But phosphatidylserine synthetase (PSS) content in intestinal bacteria low (less than the albumen total content 0.1%), be unfavorable for direct industrial fermentation production; And the subtilis expression system is a kind of comparatively ideal heterologous protein expression system, can express the heterologous protein of biologically active efficiently and it is secreted in the substratum, and have higher biological safety.The present invention adopts subtilis system expression phosphatidylserine synthetase gene, has solved the low problem of starting strain phosphatidylserine synthetase (PSS) expression amount, is applicable to suitability for industrialized production.
Phosphatidylserine synthetase has hydrolysis and transesterification double activity, and both hydrolyzable Yelkin TTS (PC) formed phosphatidic acid (PA) and choline; Again can be in the presence of nucleophiles such as high density Serine, thanomin, the catalysis transesterification reaction generates rare phosphatide---phosphatidylserine (PS), the phosphatidylethanolamine (PE) etc. with opposed polarity head.Use isotope labelling techniques the study on mechanism of enzyme is found that this enzyme shows the ping-pong reaction mechanism, the phase interface that gathers mutually at water and substrate carries out " modification " to substrate.
Phosphatidylserine (phosphatidylserine PS) is a kind of natural phosphatide, is extracted first by Jordi Folch and qualitative in addition in nineteen forty-two, and it is not a single component, but one group of compound.The structures shape of phosphatidylserine its have amphiphatic peculiar property, have an end possess hydrophilic property (or water-soluble) of negative charge, the other end of being made up of lipid acid has lipotropy (or fat-soluble).As a member in the phosphatide family, it is unique phosphatide that can regulating cell film key protein functional status, and unique physical and chemical properties and nutritive value are arranged: (1) improves cognitive ability, particularly discerns, study, memory capability; (2) improve A Erci mos disease (Alzheimer ' S Disease); (3) treatment dysthymia disorders; (4) alleviate stress and physical fatigue.
At present the main stream approach of suitability for industrialized production phosphatidylserine is to adopt extraction, exist selectivity not high, easily introduce other impurity, can't obtain pure problems such as phosphatidylserine; And chemical synthesis production phosphatidylserine can not reach the actual production requirement.Utilize the height specificity of phosphatidylserine synthetase, catalysis Yelkin TTS and Serine prepare phosphatidylserine, are expected to change at present producing the problem that exists on the phosphatidylserine, produce high-purity phospholipid class material for industrialization is directed and lay the foundation.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of selectivity height, the method for the catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase that final product impurity is few, purity is high.
The present invention is achieved through the following technical solutions:
A kind of method of catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase may further comprise the steps:
(1). the adding of phosphatidylserine synthetase enzyme liquid is contained Serine, CaCl
2, Triton-X100 acetate buffer solution in, mix with the chloroformic solution that contains Yelkin TTS then, 28～32 ℃ of condition lower magnetic force stirring reactions 3～5 hours;
(2). reaction finishes back biliquid phase natural layering, and it is standby to extract organic phase;
(3). after organic phase was volatilized naturally, residuum obtained phosphatidylserine through the chloroform washing.
And the volume ratio of described phosphatidylserine synthetase enzyme liquid and acetate buffer solution is 1: 50.
And the concentration of the Serine in the described step (1) is 3.4M.
And, the CaCl in the described step (1)
2Concentration be 20mM.
And the concentration of the Triton-X100 in the described step (1) is 0.375%.
And the pH of the acetate buffer solution in the described step (1) is 7.0.
And the concentration of the Yelkin TTS in the described step (1) is 17.0mM.
And the acetate buffer solution in the described step (1) and the volume ratio of chloroformic solution are 1: 1.
Beneficial effect of the present invention and advantage:
The present invention utilizes the height specificity of phosphatidylserine synthetase, catalysis Yelkin TTS and Serine prepare phosphatidylserine, solve present extraction and chemical synthesis effectively and can't obtain the problem of high-purity phospholipid acyl Serine, and process stabilizing is reliable, the efficient height, be fit to suitability for industrialized production, have very big application potential at medicine and health care of food product field.
Description of drawings
Fig. 1 is the pcr amplification electrophorogram of phosphatidylserine synthetase gene of the present invention;
Fig. 2 is the structure synoptic diagram of recombinant expression vector pBES-pss of the present invention;
Fig. 3 cuts proof diagram for the enzyme of recombinant expression vector pBES-pss of the present invention;
Fig. 4 is the PCR proof diagram of recombinant expression vector pBES-pss of the present invention;
Fig. 5 is the polyacrylamide gel electrophoresis figure of phosphatidylserine synthetase of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
A kind of method of catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase comprises following three partial contents, the structure of first phosphatidylserine synthetase engineering bacteria and the expression of enzyme thereof; Its two be the reorganization phosphatidylserine synthetase separation and purification; It three is reorganization phosphatidylserine synthetase transesterification synthetic phospholipid acyl Serines.Narrated respectively below:
One, the expression of the structure of phosphatidylserine synthetase engineering bacteria and enzyme thereof
The engineering bacteria that adopts is to be deposited in Chinese common micro-organisms culture presevation administrative center (CGMCC), and deposit number is the subtilis of CGMCC 1.1395, and its construction process is:
1. intestinal bacteria Escherichia coli K
12The extraction of chromosomal DNA
(1). picking intestinal bacteria Escherichia coli K
12Single colony inoculation in 5mL LB substratum, cultivate 12h in 37 ℃, 180r/min.
(2). get the 1mL nutrient solution in 1.5mL EP pipe, centrifugal 10 minutes of 12000r/min uses up supernatant, bacterial precipitation is resuspended among the alkaline lysis liquid I of 200 μ L ice precooling thermal agitation.
(3). adding 50 μ L concentration is the N,O-Diacetylmuramidase of 50mg/mL, and 4 ℃ digest 1h down.
(4). add 150 μ L concentration and be 2% SDS solution, reaction 10min presents thick to bacteria suspension.
(5). add the saturated phenol of 400 μ L: chloroform (1: 1), mix, the centrifugal 10min of 12000r/min transfers to supernatant in another clean EP pipe, discards lower floor's organic phase and albumen precipitation, repeats 1 time.
(6). add 400 μ L chloroforms, mix, centrifugal 1 0min of 12000r/min transfers to supernatant in another clean EP pipe, removes Determination of Trace Phenol.
(7). add the dehydrated alcohol deposit D NA of 800 μ L, the centrifugal 10min of 12000r/min, supernatant discarded, 500 μ L, 70% washing with alcohol 2 times.
(8). the EP pipe is inverted on the filter paper, and amount is done the back and is dissolved with the TE damping fluid, and-20 ℃ of preservations are standby.
2. design of primers and pcr amplification phosphatidylserine synthetase gene
(1) .PCR amplifying target genes: comparable data storehouse ExPASy goes up phosphatidylserine synthetase gene (pss) sequence (sequence number P23830) the design primer of report, and primer is synthetic by the handsome company in Shanghai.
Upstream primer P1:
5 '-AA
CTGCAGAATTGTCAAATTTAAGCGTAATAAAC-3 ' (underscore is the Pst I restriction enzyme site for adding partly)
Downstream primer P2:
5 '-CCC
AAGCTTTTACAGGATGCGGCTAATTAAT-3 ' (underscore is the HindIII restriction enzyme site for adding partly)
E. coli chromosomal dna (10ng) with extraction is a template, and the pcr amplification condition is: 94 ℃, and pre-sex change 5min; 94 ℃ of sex change 1min, 50 ℃ of annealing 1min40s, 72 ℃ are extended 1min, react 30 circulations; Last 72 ℃, extend 10min.Pcr amplification product downcuts the purpose band through 8g/L agarose gel electrophoresis (as Fig. 1), reclaims purifying with the DNA test kit.
(2). the extraction of large intestine-Bacillus subtilus shuttle plasmid pBES:
1.. get the Bacillus subtilis inclined-plane inoculation that an environmental protection has the pBES plasmid, line is located away from LB solid medium (the containing 30 μ g/mL Kan) flat board, is inverted for 37 ℃ and cultivates 12h.
2.. picking list bacterium colony, be linked into 5mL and contain in the LB liquid nutrient medium of Kan, cultivate 12h in 37 ℃, 180r/min.
3.. 1.5mL bacterium liquid is changed in the clean Eppendorf tube, and the centrifugal 30s of 12000r/m collects thalline, abandons supernatant.
4.. precipitation is resuspended among the alkaline lysis liquid I of 100 μ L precoolings, mixes the N,O-Diacetylmuramidase that 10 μ L50mg/mL are added in the back, 37 ℃ of insulation 30min.
5.. add 200 μ L alkaline lysis liquid II, cover the tight mouth of pipe, shake up gently, guarantee that the total inner surface bacterium of centrifuge tube contacts with alkaline lysis liquid II, centrifuge tube is placed 2min is limpid to liquid on ice.
6.. add the alkaline lysis liquid III of 150 μ L precoolings, the gentle centrifuge tube that rotates makes alkaline lysis liquid III mix ice bath 5min in the heavy-gravity bacterial lysate.
7. the centrifugal 5min of .12000r/min transfers to supernatant in another pipe, adds the saturated phenol/chloroform of Tris/primary isoamyl alcohol (25: 24: 1) mixing solutions of 400 μ L, and after mixing, the centrifugal 5min of 12000r/min transfers to supernatant in another centrifuge tube.
8.. add the dehydrated alcohol of 800 μ L, after mixing, place 30min, the centrifugal 5min collecting precipitation of 12000r/min for-20 ℃.
9.. add 1mL 70% ethanol, washing precipitation 2 times discards raffinate, and air drying 20min is with the deionized water dissolving precipitation of 20 μ L sterilization.
(3). recombinant vectors pBES-pss makes up: the plasmid pBES of PCR product and extraction is carried out double digestion with Pst I and HindIII respectively, after enzyme is cut the purified recovery of product, spend the night in 16 ℃ of connections with Solution I and to obtain recombinant plasmid pBES-pss, plasmid construction such as Fig. 2.
3. the conversion of recombinant vectors and the expression in host bacteria
(1). the conversion of recombinant vectors and evaluation: subtilis transforms and improves on the basis of reference Spizizen method.Get subtilis DB104 and draw the LA flat board, 37 ℃ of incubators are cultivated 12h.Choose single bacterium colony to 3mL SPI substratum, 37 ℃, 250r/min are cultivated 12h.Get 100 μ L nutrient solutions next day and be forwarded to 5mL SPI substratum, 37 ℃, 250r/min are cultured to logarithmic growth latter stage (about 4h, OD
600Be about 0.9), get the 0.2mL nutrient solution to 2mL SPII substratum, 37 ℃, 150r/min are cultivated 90min.Be distributed into the every pipe of 0.5mL, each adds 20 μ L recombinant plasmids, and in 37 ℃, 100r/min concussion cultivation 30min, 250r/min cultivates 90min then again, and centrifugal place to go supernatant liquor keeps 100 μ L supernatant liquors and bacterium liquid and mixes back coating screening flat board.
Utilize the Spizizen method that recombinant plasmid is changed among the subtilis DB104, by kalamycin resistance plate screening transformant, enzyme is cut with PCR and is verified the result as shown in Figure 3 and Figure 4, shows correctly to be connected with goal gene on the recombinant plasmid.
(2). the abduction delivering of reorganization bacterium: inoculation recombination bacillus subtilis DB104/pBES-pss and contrast bacterium DB104/pBES contain in the LB substratum of 30 μ g/mL kantlex 37 ℃ of shaking culture 12h in 5mL.Inoculum size switching overnight culture by 5% contains in the LB substratum of 30 μ g/mL kantlex in 30mL, and adding 20% sucrose solution to final concentration behind the shaking culture 2h is 2%, induces 24h.After fermented liquid is centrifugal, respectively get reorganization bacterium and contrast bacterium supernatant 10 μ L, mix with 10 μ L sample-loading buffers respectively, boiling water bath 10min, the centrifuging and taking supernatant carries out SDS-PAGE (12% separation gel, 5% concentrates glue) electrophoresis, detect protein expression situation such as Fig. 5, fermented liquid supernatant can be directly used in enzyme activity determination.
(3). the enzyme biopsy is surveyed: adopt enzyme connection colorimetry to carry out activity and detect.
Phosphatidylserine synthetase (PSS) 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, have light absorption value under A=500nm.
Reaction system: the mixed system of ethanolic soln (dense eventually is 1%) of deionized water, 0.272mL 17.9% 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 peroxidase 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 35 ℃ of water-baths.The enzyme liquid that adds 0.1mL then, mix, in 35 ℃ of reaction 10min, the boiling water bath termination reaction, to be cooled to room temperature adding 0.05mL 2M TrisHCl (pH=9) damping fluid, after mixing was also centrifugal, supernatant was got filtrate 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.Add the 2.0mL deionized water in reaction mixture, centrifugal obtaining clarified bright pink solution, at last in A
500nmDetect light absorption value.
Enzyme is lived and defined: in the time of pH=8.0, T=35 ℃, catalytic hydrolysis L-α-Yelkin TTS discharges the needed enzyme amount of choline of 1.0 μ mol in the phosphatidylserine synthetase 1h.
Two, the separation and purification of reorganization phosphatidylserine synthetase
1. prepare thick fermented liquid
Engineering bacteria is seeded in the LB liquid nutrient medium, 37 ℃ of shaking culture 12h, the cultivation after product is forwarded to continuation cultivation 2h in the same medium by 5% inoculum size, add 10% aqueous sucrose solution then, induce 28h, induce after product centrifugal 20min under 4 ℃, the condition of 8000r/min, collect supernatant, make thick fermented liquid.
2. saltout
Get the 28h fermented liquid, centrifugal 20min under 4 ℃, the condition of 8000r/min collects supernatant, add ammonium sulfate to 75% saturation ratio, 4 ℃ leave standstill 12h after, centrifugal 20min under 4 ℃, the condition of 8000r/min, the supernatant that inclines, precipitation is dissolved in the Tris-HCl damping fluid of pH 7.0,10mM.
3. desalination
After salt precipitation being dissolved in the Tris-HCl damping fluid of pH 7.0,10mM, be the tubular fibre membrane ultrafiltration of 10kDa～20kDa, sample carried out desalination and concentration, desalination effect BaCl with the molecular weight that dams
2Solution detects, and splashes into BaCl with filtrate
2Do not have in the solution to precipitate to separate out and be terminal point.
4.SP-Sepharose the HP (ion exchange chromatography of 2.4cm * 30cm)
Adopt the Tris-HCl damping fluid balance chromatography column of pH 7.0,10mM, active ingredient after the desalination is dissolved with same damping fluid, the centrifugal 20min of 8000r/min, get supernatant, the albumen that does not adsorb with this buffer solution elution earlier behind the last sample 2mL, carry out gradient elution with this damping fluid that contains 0～1mol/L NaCl again, collect active ingredient.
5.Sephadex the G-75 (gel chromatography of 1.6cm * 80cm)
The active ingredient that ion exchange chromatography obtains, earlier with the Tris-HCl damping fluid dissolving that contains 1.0%NaCl 10mM, the centrifugal 20min of 8000r/min, behind the last sample 2mL with the speed wash-out of identical damping fluid with 0.5mL/min, every pipe is collected 2.0mL, collect active ingredient, be phosphatidylserine synthetase.
Three, reorganization phosphatidylserine synthetase transesterification synthetic phospholipid acyl Serine
Biliquid phase reaction system: by 1: 50 volume ratio the phosphatidylserine synthetase enzyme liquid of purifying is added and to contain 3.4M Serine, 20mM CaCl
2, 0.375%Triton-X100 acetate buffer solution (pH=7.0) in, mix with the chloroformic solution equal-volume that contains 17.0mM Yelkin TTS then, mix at 30 ℃ of condition lower magnetic forces and obtain uniform milk sap and react.Behind the reaction 5h, stop to stir biliquid phase natural layering.Extract organic phase, after organic phase was volatilized naturally, residuum can obtain highly purified phosphatidylserine through the chloroform washed twice.At last product is dissolved in an amount of chloroform: in the methyl alcohol=19: 1 (v/v), with usefulness to be analyzed.
Adopt HPLC to detect the transformation efficiency of phosphatidylserine, the chromatographic condition of detection is:
Detect wavelength: 206nm;
Moving phase: acetonitrile: methyl alcohol: 85% phosphoric acid=180: 3: 1;
Chromatographic column: Kromasil SiO
25 μ (250 * 4.6mm);
Flow velocity: 0.5mL/min;
Sample size: 10 μ L;
Detection time: 15min.
It is an amount of to take by weighing the phosphatidylserine standard substance respectively, be made into chloroform and methyl alcohol (volume ratio 19: 1) that concentration is respectively 0.2,0.5,1.0,1.5,2.0, the standardized solution of 2.5mg/mL, (mg/mL) is X-coordinate with concentration of standard solution, is ordinate zou with the peak area, the drawing curve.Utilize the standard working curve of phosphatidylserine, calculate the product growing amount, thereby draw the transformation efficiency result.
SEQUENCE?LISTING
＜110〉University Of Science and Technology Of Tianjin
＜120〉the efficient construction process of the engineering bacteria of heterogenous expression phosphatidylserine synthetase
<130>20080703
<160>2
<170>PatentIn?version?3.3
<210>1
<211>1356
<212>DNA
＜213〉phosphatidylserine synthetase (phosphatidylserine synthase, dna sequence dna PSS)
<400>1
atgttgtcaa?aatttaagcg?taataaacat?caacaacacc?ttgcccaact?acccaagatt 60
tctcaatcag?ttgatgatgt?cgatttcttt?tacgctcccg?ccgacttccg?ggagacgctg 120
ctggaaaaaa?tagccagcgc?gaagcagcgc?atttgcattg?tcgccctgta?tctcgaacag 180
gatgacggtg?gcaaaggcat?tctgaacgcg?ttgtatgagg?ctaaaaggca?ggacgatccg 240
gaactggatg?tgcgggtgct?ggtcgactgg?catcgtgcac?aacgtggacg?cattggcgct 300
gcggcatcta?acactaacgc?tgactggtac?tgccgcatgg?cgcaggaaaa?tccgggcgta 360
gatgttccgg?tttatggcgt?tccaatcaat?actcgtgaag?cccttggtgt?tctgcacttt 420
aaaggcttta?tcatcgacga?tagcgtactt?tatagcggtg?ccagcctgaa?cgatgtttac 480
ctgcatcagc?acgataaata?tcgctacgac?cgttatcatc?tgatccgtaa?ccgtaagatg 540
tcagacatta?tgtttgaatg?ggttacacag?aatattatga?atggccgcgg?cgttaatcgt 600
ctggatgatg?ttaatcggcc?aaaaagcccg?gaaatcaaga?acgatattcg?tctgttccgc 660
caggagctgc?gtgatgccgc?ttatcatttc?cagggcgatg?ccgacaacga?tcagctttct 720
gtaacgccgc?tagtggggct?ggggaaatcg?agtctgttga?acaagaccat?tttccatctt 780
atgccttgtg?ccgagcagaa?actaaccatc?tgtacgccat?acttcaacct?gccagcaatc 840
cttgtgcgca?atattatcca?gttgctgcgc?gaagggaaaa?aggtcgaaat?tattgttggt 900
gataaaaccg?cgaatgactt?ctacattccg?gaagatgaac?ctttcaagat?aattggcgca 960
ttgccttatc?tctatgagat?caatttgcgt?cgtttcctga?gccgtttgca?gtattacgtc 1020
aatactgacc?agctagtggt?tcggttatgg?aaagatgacg?acaacaccta?tcacctgaaa 1080
gggatgtggg?ttgatgataa?gtggatgttg?atcaccggta?ataacctgaa?cccgcgcgcc 1140
tggcgtctgg?atctggaaaa?cgccattttg?atccacgatc?cgcaacttga?gctggcgcca 1200
cagcgagaga?aagaactgga?gctgatccgc?gagcatacca?ccatcgttaa?gcactatcgc 1260
gatctgcaaa?gtattgccga?ttatccggtg?aaggttcgta?aactcatccg?ccgttcgcgc 1320
cgtatccgca?tcgaccgatt?aattagccgc?atcctg 1356
<210>2
<211>451
<212>PRT
＜213〉phosphatidylserine synthetase (phosphatidylserine synthase, aminoacid sequence PSS)
<400>2
Met?Leu?Ser?Lys?Phe?Lys?Arg?Asn?Lys?His?Gln?Gln?His?Leu?Ala?Gln
1 5 10 15
Leu?Pro?Lys?Ile?Ser?Gln?Ser?Val?Asp?Asp?Val?Asp?Phe?Phe?Tyr?Ala
20 25 30
Pro?Ala?Asp?Phe?Arg?Glu?Thr?Leu?Leu?Glu?Lys?Ile?Ala?Ser?Ala?Lys
35 40 45
Gln?Arg?Ile?Cys?Ile?Val?Ala?Leu?Tyr?Leu?Glu?Gln?Asp?Asp?Gly?Gly
50 55 60
Lys?Gly?Ile?Leu?Asn?Ala?Leu?Tyr?Glu?Ala?Lys?Arg?Gln?Arg?Pro?Glu
65 70 75 80
Leu?Asp?Val?Arg?Val?Leu?Val?Asp?Trp?His?Arg?Ala?Gln?Arg?Gly?Arg
85 90 95
Ile?Gly?Ala?Ala?Ala?Ser?Asn?Thr?Asn?Ala?Asp?Trp?Tyr?Cys?Arg?Met
100 105 110
Ala?Gln?Glu?Asn?Pro?Gly?Val?Asp?Val?Pro?Val?Tyr?Gly?Val?Pro?Ile
115 120 125
Asn?Thr?Arg?Glu?Ala?Leu?Gly?Val?Leu?His?Phe?Lys?Gly?Phe?Ile?Ile
130 135 140
Asp?Asp?Ser?Val?Leu?Tyr?Ser?Gly?Ala?Ser?Leu?Asn?Asp?Val?Tyr?Leu
145 150 155 160
His?Gln?His?Asp?Lys?Tyr?Arg?Tyr?Asp?Arg?Tyr?His?Leu?Ile?Arg?Asn
165 170 175
Arg?Lys?Met?Ser?Asp?Ile?Met?Phe?Glu?Trp?Val?Thr?Gln?Asn?Ile?Met
180 185 190
Asn?Gly?Arg?Gly?Val?Asn?Arg?Leu?Asp?Asp?Val?Asn?Arg?Pro?Lys?Ser
195 200 205
Pro?Glu?Ile?Lys?Asn?Asp?Ile?Arg?Leu?Phe?Arg?Gln?Glu?Leu?Arg?Asp
210 215 220
Ala?Ala?Tyr?His?Phe?Gln?Gly?Asp?Ala?Asp?Asn?Asp?Gln?Leu?Ser?Val
225 230 235 240
Thr?Pro?Leu?Val?Gly?Leu?Gly?Lys?Ser?Ser?Leu?Leu?Asn?Lys?Thr?Ile
245 250 255
Phe?His?Leu?Met?Pro?Cys?Ala?Glu?Gln?Lys?Leu?Thr?Ile?Cys?Thr?Pro
260 265 270
Tyr?Phe?Asn?Leu?Pro?Ala?Ile?Leu?Val?Arg?Asn?Ile?Ile?Gln?Leu?Leu
275 280 285
Arg?Glu?Gly?Lys?Lys?Val?Glu?Ile?Ile?Val?Gly?Asp?Lys?Thr?Ala?Asn
290 295 300
Asp?Phe?Tyr?Ile?Pro?Glu?Asp?Glu?Pro?Phe?Lys?Ile?Ile?Gly?Ala?Leu
305 310 315 320
Pro?Tyr?Leu?Tyr?Glu?Ile?Asn?Leu?Arg?Arg?Phe?Leu?Ser?Arg?Leu?Gln
325 330 335
Tyr?Tyr?Val?Asn?Thr?Asp?Gln?Leu?Val?Val?Arg?Leu?Trp?Lys?Asp?Asp
340 345 350
Asp?Asn?Thr?Tyr?His?Leu?Lys?Gly?Met?Trp?Val?Asp?Asp?Lys?Trp?Met
355 360 365
Leu?Ile?Thr?Gly?Asn?Asn?Leu?Asn?Pro?Arg?Ala?Trp?Arg?Leu?Asp?Leu
370 375 380
Glu?Asn?Ala?Ile?Leu?Ile?His?Asp?Pro?Gln?Leu?Glu?Leu?Ala?Pro?Gln
385 390 395 400
Arg?Glu?Lys?Glu?Leu?Glu?Leu?Ile?Arg?Glu?His?Thr?Thr?Ile?Val?Lys
405 410 415
His?Tyr?Arg?Asp?Leu?Gln?Ser?Ile?Ala?Asp?Tyr?Pro?Val?Lys?Val?Arg
420 425 430
Lys?Leu?Ile?Arg?Arg?Leu?Arg?Arg?Ile?Arg?Ile?Asp?Arg?Leu?Ile?Ser
435 440 445
Arg?Ile?Leu
450
Claims (8)
1, a kind of method of catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase is characterized in that: may further comprise the steps:
(1). the adding of phosphatidylserine synthetase enzyme liquid is contained Serine, CaCl
2, Triton-X100 acetate buffer solution in, mix with the chloroformic solution that contains Yelkin TTS then, 28～32 ℃ of condition lower magnetic force stirring reactions 3～5 hours;
(2). reaction finishes back biliquid phase natural layering, and it is standby to extract organic phase;
(3). after organic phase was volatilized naturally, residuum obtained phosphatidylserine through the chloroform washing.
2, the method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase according to claim 1 is characterized in that: the volume ratio of described phosphatidylserine synthetase enzyme liquid and acetate buffer solution is 1: 50.
3, the method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase according to claim 1 is characterized in that: the concentration of the Serine in the described step (1) is 3.4M.
4, the method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase according to claim 1 is characterized in that: the CaCl in the described step (1)
2Concentration be 20mM.
5, the method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase according to claim 1 is characterized in that: the concentration of the Triton-X100 in the described step (1) is 0.375%.
6, the method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase according to claim 1 is characterized in that: the pH of the acetate buffer solution in the described step (1) is 7.0.
7, the method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase according to claim 1 is characterized in that: the concentration of the Yelkin TTS in the described step (1) is 17.0mM.
8, the method for catalysis synthesis of phosphatidylserine with phosphatidylserine synthetase according to claim 1 is characterized in that: the acetate buffer solution in the described step (1) and the volume ratio of chloroformic solution are 1: 1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277394A (en) * | 2011-07-22 | 2011-12-14 | 天津科技大学 | Method for preparing phosphatidylserine in presence of cell-surface display phospholipase D yeast whole cell catalyst |
| CN103884813A (en) * | 2014-04-11 | 2014-06-25 | 内蒙古伊利实业集团股份有限公司 | Method for detecting content of phosphatidylserine in food |
| CN105567755A (en) * | 2016-01-28 | 2016-05-11 | 成都显诚生物工程有限公司 | Preparation method of phosphatidylserine |
| CN107727755A (en) * | 2017-09-06 | 2018-02-23 | 南通励成生物工程有限公司 | A kind of detection method of phosphatidylserine |
| CN112352051A (en) * | 2019-12-31 | 2021-02-09 | 邦泰生物工程(深圳)有限公司 | Method for improving phospholipase D (phospholipase D) translipidation activity and method for producing phosphatidylserine by using phospholipase D translipidation activity |
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2008
- 2008-07-11 CN CNA2008100538127A patent/CN101319237A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277394A (en) * | 2011-07-22 | 2011-12-14 | 天津科技大学 | Method for preparing phosphatidylserine in presence of cell-surface display phospholipase D yeast whole cell catalyst |
| CN102277394B (en) * | 2011-07-22 | 2013-04-17 | 天津科技大学 | Method for preparing phosphatidylserine in presence of cell-surface display phospholipase D yeast whole cell catalyst |
| CN103884813A (en) * | 2014-04-11 | 2014-06-25 | 内蒙古伊利实业集团股份有限公司 | Method for detecting content of phosphatidylserine in food |
| CN105567755A (en) * | 2016-01-28 | 2016-05-11 | 成都显诚生物工程有限公司 | Preparation method of phosphatidylserine |
| CN107727755A (en) * | 2017-09-06 | 2018-02-23 | 南通励成生物工程有限公司 | A kind of detection method of phosphatidylserine |
| CN112352051A (en) * | 2019-12-31 | 2021-02-09 | 邦泰生物工程(深圳)有限公司 | Method for improving phospholipase D (phospholipase D) translipidation activity and method for producing phosphatidylserine by using phospholipase D translipidation activity |
| WO2021134439A1 (en) * | 2019-12-31 | 2021-07-08 | 邦泰生物工程(深圳)有限公司 | Method for improving transesterification activity of phospholipase d and method for producing phosphatidylserine using phospholipase d |
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