CN107164397A - The gene excavating and its application process of a kind of phospholipase D - Google Patents
The gene excavating and its application process of a kind of phospholipase D Download PDFInfo
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Abstract
The invention provides a kind of phospholipase D and its gene coded sequence and application process obtained based on gene excavating method.The phospholipase D total length 1,614bp, encodes 538 amino acid, with plasmid pET28a (+) for expression vector, with E. coli BL21 (DE3) for expressive host, realizes the high efficient expression of phospholipase D.The gene excavating method of the phospholipase D is simply convenient, can secrete phospholipase D through excavating the fermented induction of recombinant bacterial strain constructed by the target gene obtained, the optimal reactive temperature of the phospholipase D is 60 DEG C, and optimal reaction pH is 7.5, and Ca2+There is preferably activation facilitation to its enzyme activity.The enzyme can be modified to phosphatide, catalysis substrate phosphatidyl choline and L serine synthetic phospholipid acyl serines, be had a good application prospect in food, medicine and healthcare industry.
Description
Technical field
The invention provides a kind of phospholipase D and its application process obtained based on database gene excavating method, belong to
Industrial biotechnology field.
Background technology
Phospholipase D (PLD) is the general name for the enzyme that class catalytic phosphatase diester linkage hydrolysis and Baseexchange react, main substrate
It is phosphatidyl choline (PC), PLD can be by PC hydrolysis generation phosphatidic acids (PA) and choline, and turning phosphatidyl vigor using it can make
Standby many functions good rare phosphatide and phospholipid derivative, are widely used in food, medicine and field of health care products.
Phospholipase D wide material sources, report that it is derived from Chinese cabbage leaf, then in other animal and plant and microorganism earliest
It was found that, the medium clone of yeast, bacterium, which obtains PLD, also relevant report.Phospholipase D is primarily present in seed, root and leaf in plant
In；Animal origin is mainly distributed in brain, liver.Compared with the phospholipase D in other sources, Microbial phospholipases D has stronger
Substrate tolerance, wider Substratspezifitaet and higher Baseexchange catalytic reaction activity, more suitable for commercial Application.Extremely
The microorganism for the production phospholipase D that the present has been reported mainly has streptomycete, corynebacteria, Escherichia coli, Salmonella and false list
Spore bacterium etc..Wherein, phospholipase D is distributed the most extensively in streptomycete, reports also most.
The external research to PLD is constantly in top standard, and successful foreign is screened and utilizes the methods such as genetic recombination to transform
Go out high yield PLD biological bacterial strain, beneficial to industrial production, a small number of enzyme preparation companies have developed the PLD with high enzyme vigor,
Domestic that the research that microbial fermentation produces PLD is started late, enzyme activity is relatively low, and import is relied primarily at present, but price is very expensive.
PLD enzyme preparation of the research with independent intellectual property right has realistic meaning, and bacterial strain screening is one of important step.
Phosphatidylserine (PS) is one kind of phosphatide, and natural content is less, but it is main acidic phospholipid in brain,
It can control and adjust the functional status of cell membrane key protein.PS accounts for the 10%-20% of cell membrane bilayer phosphatide, to maincenter
Effects on neural system significantly, can improve the vigor of brain cell, improve cerebral function, brain damage repaired, as " brain selectivity nutrients
Matter ", gradually causes people's extensive concern.Phosphatidylserine preparation method mainly includes extraction method, chemical synthesis and biology
Enzyme transforming process.Extraction method refers mainly to directly extract phosphatidylserine from plant cell, the brain of animal and internal organ.Correlation is ground
The content for studying carefully phosphatidylserine in display plant tissue is considerably less, and extraction method is mainly with animal tissue such as brain and internal organ etc.
It is main.Extraction method is extracted using organic solvent, simple to operate, but purity and low yield, and pollution is big, and due to rabid ox disease
Reason, the PS of extraction also has certain safety issue；Chemical synthesis is referred mainly to by passing through one by primary raw material of glycerine
Sequence of chemical reaction synthesis PS, product purity that the method is obtained is high, and security is good, but synthesis step is cumbersome and accessory substance compared with
It is many.In recent years, bioenzymatic conversion method is gradually paid attention to by everybody.Enzyme transforming process refers under the catalytic action of phospholipase D, bottom
Thing lecithin and serine carry out transacylate reaction, generate phosphatidylserine.This method has mild condition, production cost
Low, accessory substance is few, low-carbon environment-friendly, the advantages of yield is high, is the Perfected process for preparing a large amount of phosphatidylserines.Although, it is domestic
The method of Enzyme catalyzed synthesis phosphatidylserine is reported outside, but is mostly wild mushroom, fermentation time is long, and to the property of enzyme
Not enough understand, so as to have impact on the efficiency of Enzyme catalyzed synthesis phosphatidylserine, therefore the clonal expression of phospholipase D and its
High efficient expression in Escherichia coli, seems extremely important to its practical application and Study on mechanism.
The content of the invention
It is an object of the invention to provide a kind of phospholipase D obtained based on gene excavating and its gene coded sequence and application
Method.Offer includes the plasmid of gene of the present invention and the host cell comprising the expression plasmid, and utilizes the recombinant bacterial strain table
Up to the method for production phospholipase D.
Phospholipase D is obtained by database gene excavating technology screening.To turn phosphatidyl vigor phosphatide with higher
Enzyme D amino acid sequence is template, and BLAST is carried out in ncbi database and compares analysis, according to the screening criteria of setting, is obtained
Purpose phospholipase D, designs primer according to gene order and passes through round pcr amplification acquisition coding gene sequence, electroresis appraisal
Recovery purifying PCR primer and pMD19-T vector construction recombinant plasmids are cloned into afterwards, by recombinant plasmid transformed to E.coli
JM109, the multiple positive transformants of picking (Amp into LB fluid nutrient mediumsr) 37 DEG C, 220rpm cultivates 10~12h, extracts plasmid
Double digestion, PCR checkings are carried out afterwards.It will verify that correct recombinant plasmid is same double with warp after BamH I and Hind III double digestions
The vector plasmid of digestion is attached, and the vector plasmid of selection includes pET series, and pMA5, pWB is serial, pPICZ α, PIC9K,
PDW series etc..Connection product is converted into Host Strains large intestine competent cell, but is not limited to Escherichia coli, in addition to thread
Fungi, bacillus subtilis, Corynebacterium glutamicum, Corynebacterium crenatum, Pichia pastoris, saccharomyces cerevisiae etc., phosphorus is realized after culture
The high efficient expression of lipase D.
(1) database gene excavating technology screening phospholipase D
According to phospholipase D heterogenous expression document has been reported, selection turns the amino acid sequence of the higher phospholipase D of phosphatidyl vigor
Row are as template, the BLAST in ncbi database, and a series of phospholipase D amino acid sequences are obtained according to the screening criteria of setting,
The amino acid sequence of purpose phospholipase D is obtained by building phylogenetic analysis screening again, so as to obtain its coding gene sequence.
(2) PCR expands phospholipase D
With the nucleotide sequence design primer (P1, P2) through synthesis, the encoding gene of phospholipase D is expanded by PCR：
Primer P1：5’-CGGGA TCC ATG CTG CGT CGC CTG CAT
Primer P2：5’-CCCAAG CTT TTA CAG ACT ACA CAC ACC
Pcr amplification reaction is carried out in 50 μ L systems, and 25 μ L Ex Taq (Premix), 20 μ L are added in reaction system
ddH2O, 2 μ L template DNAs, each 1.5 μ L of upstream and downstream primer.Reaction condition is to be started the cycle over after 94 DEG C of pre-degeneration 3min：94℃
It is denatured 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 circulations；72 DEG C extend 10min eventually.PCR primer carries out nov nucleic acid
Swimming identification and recovery purifying of tapping rubber, recovery product is cloned into pMD19-T vector construction recombinant clone plasmids, by plasmid convert to
E.coli JM109, the multiple positive transformants of picking (Amp into LB fluid nutrient mediumsr) 37 DEG C, 220rpm cultivates 10~12h,
Extract and carry out double digestion, PCR checkings after plasmid to plasmid, checking is correct to carry out sequencing.
(3) structure of recombinant expression plasmid
The structure of recombinant expression plasmid is carried out in this research by taking pET-28a (+) as an example, the plasmid with T7 promoters and 6 ×
His-tag labels, BamH is used by pET-28a (+) plasmid and the correct recombinant clone plasmid containing target gene of checking simultaneously
I and Hind III carry out carrying out nucleic acid electrophoresis checking after double digestion, 37 DEG C of digestion 3h and tap rubber recovery target gene and purpose matter
Grain, recovery product is connected overnight with T4DNA ligases at 16 DEG C, and connection product is converted to E.coli JM109 competent cells
In, 8~10h of culture in the LB solid cultures containing kalamycin resistance (50mg/mL) is coated on, the multiple positive transformants of picking are extremely
Cultivated in LB fluid nutrient mediums containing kanamycins (50mg/mL), cultivate after 10~12h and extract at 37 DEG C, under the conditions of 220rpm
Plasmid is simultaneously named as pET-28a (+)-pld.
(4) structure of recombinant strains and screening
This research by taking Escherichia coli as an example, research by recombinant plasmid pET-28a (+)-pld through 42 DEG C of thermal shock 90s convert to
In escherichia coli host E.coli BL21 (DE3) competent cell, the LB solids containing kanamycins (50mg/mL) are coated on
8~10h is cultivated on culture medium, by PCR checking screening positive transformant E.coli BL21 (DE3)/pET-28a (+)-pld,
It is seeded to overnight incubation in the LB fluid nutrient mediums containing kanamycins (50mg/mL).Next day is inoculated in contain by 1% inoculum concentration blocks that
In the fresh LB fluid nutrient mediums of 50mL of mycin (50mg/mL), 37 DEG C of culture about 3h add IPTG and carried out when OD600 is 1.0
Induction, makes after its final concentration of 0.5mM, 25 DEG C of induction 12h, recombinant bacterium shows phospholipase D activity.
(5) the Western Blot analyses of restructuring phospholipase D
By the restructuring fermented liquid of above-mentioned acquisition at 4 DEG C, 20min is centrifuged under the conditions of 8,000rpm, removes supernatant and collects bacterium
Body is precipitated, and thalline goes the removal of impurity one time with 5ml Tris-HCl (pH7.5) buffer solution for cleaning, and 8,000rpm centrifuge 10min, again
Remove supernatant and collect bacterial sediment, suspended with 5mL Tris-HCl (pH7.5) buffer solution, be prepared into bacteria suspension.Bacteria suspension is existed
It is placed in ice-water bath and carries out ultrasonication using cell Ultrasonic Cell Disruptor, condition is：Work 200 and circulate under 200W power, often
Individual circulation work 4s stops 6s.At 4 DEG C after crushing completely, 20min is centrifuged under the conditions of 12,000rpm, gained supernatant is crude enzyme liquid.
Pass through BCA protein quantification kit measurement protein contents.After preparative separation glue and concentration glue, 80 μ L samples are taken to add 20 μ L
5 x loading buffer, fully mix makes albuminous degeneration after placement 5min in boiling water bath.According to the concentration for determining albumen
Loading is carried out, and adds Marker.Upper strata concentrates glue 80V voltages, when sample to separation gel (about 30min), with 100V electricity
Pressure, closes power supply at sample to separation gel bottom about 1cm, and electrophoresis terminates.It is clean with distilled water flushing after end, make in electric current
Under, protein is set to be transferred to from glue on solid phase carrier PVDF (PVDF membrane).Transfer is closed after finishing, and is incubated
Primary antibody and secondary antibody, finally carry out colour developing and take pictures to confirm the expression of destination protein.
(6) zymologic property research of phospholipase D is recombinated
A. phospholipase D optimal pH and pH stability studies are recombinated
Appropriate enzyme liquid is taken, respectively in 40mM pH 4.0-6.0 sodium acetate buffer, pH 6.0-8.0 phosphate-buffered
(unit of pH intervals 0.5) is reacted in liquid, pH 8.0-10 Tris-HCl buffer solutions, determines the enzyme activity under different pH, with
Maximum enzyme activity is 100%, to determine the most suitable action pH of the enzyme.Separately take second of the fresh enzyme liquid respectively in 40mM pH 4.0-6.0
(pH intervals 0.5 are single in sour sodium buffer solution, pH 6.0-8.0 phosphate buffer, pH 8.0-10 Tris-HCl buffer solutions
Position) 60 min are placed, pull back to after pH 8.0, the mode according still further to standard test enzyme activity determines remnant enzyme activity, to determine the enzyme
PH stability, be used as 100% using undressed enzyme liquid.
B. phospholipase D optimum temperature and temperature stabilization Journal of Sex Research are recombinated
Appropriate enzyme liquid is taken, temperature spot (5 DEG C of interval) is selected in the range of 20 DEG C to 80 DEG C respectively, determines it in different temperatures
Under enzyme activity, using maximum enzyme activity as 100%, to determine the optimum temperature of the enzyme.Separately take new enzyme liquid, be incubated respectively in
Cooled down immediately after 60min in above-mentioned different temperatures, remaining enzyme activity is determined in the way of standard test enzyme activity, to determine the enzyme
Temperature stability, 100% is used as using undressed enzyme liquid.
C. influence of the metal ion to restructuring phospholipase D enzyme activity
Take KCl, MgCl2、BaCl2、CaCl2、CoCl2、NaCl、CuCl2、MnCl2、LiCl、FeCl3、FeCl2、AgNO3、
ZnSO4、Al2(SO4)3Etc. 50mM mother liquors are configured to, the mother liquor prepared is separately added into the enzyme liquid suitably diluted, it is final concentration of
5mM.After 37 DEG C of insulation 30min, remnant enzyme activity is determined according to enzyme activity determination method, is made with the enzyme liquid vigor for being not added with metal ion
For 100%.
D. influence of the protein inhibitor to restructuring phospholipase D enzyme activity
Take protein inhibitor：Beta -mercaptoethanol, DTT, PMSF, EDTA, SDS etc. are configured to 50mM mother liquors, are suitably diluting
Enzyme liquid in be separately added into the mother liquor prepared, final concentration of 5mM.After 37 DEG C of insulation 30min, determine residual according to enzyme activity determination method
Remaining enzyme activity, using untreated enzyme liquid vigor as 100%.
(7) application of the restructuring phospholipase D in phosphatidylserine is catalyzed and synthesized
Include organic phase and aqueous phase two parts using the reaction system for catalyzing and synthesizing phosphatidylserine for recombinating phosphatidase,
Organic phase：Ethyl acetate of the 8mL dissolved with 10mg/mLPC60；Aqueous phase：4mL is dissolved with 30mg/mL Serines, 15mM CaCl2
Crude enzyme liquid, organic phase and aqueous phase are sufficiently mixed uniform after 40 DEG C, 10h are reacted under the conditions of 180rpm.Detected by HPLC
The synthesis of phosphatidylserine in product.
The advantages of the present invention
The invention provides a kind of phospholipase D and its high efficient expression, it has SEQ ID NO:Nucleotides shown in 1
Sequence and SEQ ID NO：Amino acid sequence shown in 2, total length 1,614bp encodes 538 amino acid, with plasmid pET28a (+)
For expression vector, with E. coli BL21 (DE3) for expressive host, the high efficient expression of phospholipase D is realized,
Recombinant bacterial strain E.coli BL21 (DE3)/pET-28a (+)-pld shows phospholipase D activity, can be catalyzed substrate phosphatidyl choline
With Serine synthetic phospholipid acyl serine, there is larger application prospect, and recombinant bacterium in food, medicine and healthcare industry
Fermentation period is short, is suitable for industrializing large-scale production.
Brief description of the drawings
Fig. 1 PCR expand phospholipase D encoding gene nucleic acid electrophoresis collection of illustrative plates
M：5,000bp DNA Marker；Swimming lane 1：Expand obtained phospholipase D.
The Western Blot analyses of Fig. 2 restructuring phospholipase Ds
M：Standard protein molecular weight；Diagram SMPLD is purpose protein, phospholipid enzyme D.
The optimal pH and pH stability of Fig. 3 phospholipase Ds
The optimum temperature and temperature stability of Fig. 4 phospholipase Ds
Fig. 5 phospholipase Ds catalyze and synthesize the HPLC testing results of phosphatidylserine
A：PS standard specimens；B：PS samples.
Embodiment
With reference to embodiment, the invention will be further described, but the present invention is not limited to these embodiments restrictions.
Embodiment 1
This example illustrates the method based on database gene excavating technology screening phospholipase D.
According to phospholipase D heterogenous expression document has been reported, select experiments verify that crosses turns the higher phosphorus of phosphatidyl vigor
The amino acid sequence of lipase D carries out BLAST as template in ncbi database, is sieved according to the screening criteria set
Choosing, obtains a series of phospholipase D amino acid sequences, then by building the amino of phylogenetic analysis screening acquisition purpose phospholipase D
Acid sequence, so as to obtain its coding gene sequence.
Embodiment 2
This example demonstrates that the cloning process of phospholipase D encoding gene.
With the nucleotide sequence design primer (P1, P2) through synthesis, the encoding gene of phospholipase D is expanded by PCR：
Primer P1：5’-CGGGA TCC ATG CTG CGT CGC CTG CAT
Primer P2：5’-CCCAAG CTT TTA CAG ACT ACA CAC ACC
Pcr amplification reaction is carried out in 50 μ L systems, and 25 μ L Ex Taq (Premix), 20 μ L are added in reaction system
ddH2O, 2 μ L template DNAs, each 1.5 μ L of upstream and downstream primer.Reaction condition is to be started the cycle over after 94 DEG C of pre-degeneration 3min：94℃
It is denatured 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 30 circulations；72 DEG C extend 10min eventually.PCR primer carries out nov nucleic acid
Swimming identification and recovery purifying of tapping rubber, recovery product is cloned into pMD19-T vector construction recombinant clone plasmids, by plasmid convert to
E.coli JM109, the multiple positive transformants of picking (Amp into LB fluid nutrient mediumsr) 37 DEG C, 220rpm cultivates 10~12h,
Extract and carry out double digestion, PCR checkings after plasmid to plasmid, checking is correct to carry out sequencing.Nucleic acid electrophoresis result shows phosphorus
Lipase D gene order obtains effectively expanding (Fig. 1).
Embodiment 3
The present embodiment illustrates the building process of recombinant expression plasmid by taking pET-28a (+) as an example.
PET-28a (+) carries T7 promoters and 6 × His-tag labels, and pET-28a (+) plasmid and checking are correctly contained
The recombinant clone plasmid of purposeful gene carries out nucleic acid after double digestion, 37 DEG C of digestion 3h are carried out with BamH I and Hind III simultaneously
Electrophoresis verifies and tap rubber recovery target gene and purpose plasmid that recovery product is connected overnight with T4DNA ligases at 16 DEG C, connection
Product is converted into E.coli JM109 competent cells, is coated on the LB solid cultures containing kalamycin resistance (50mg/mL)
8~10h of middle culture, the multiple positive transformants of picking are cultivated into the LB fluid nutrient mediums containing kanamycins (50mg/mL), 37
DEG C, extract plasmid after 10~12h of culture under the conditions of 220rpm and be named as pET-28a (+)-pld.
Embodiment 4
The present embodiment illustrates the structure and screening technique of recombinant strains by taking Escherichia coli as an example.
Recombinant plasmid pET-28a (+)-pld are converted to escherichia coli host E.coli BL21 through 42 DEG C of thermal shock 90s
(DE3) in competent cell, 8~10h of culture on the LB solid mediums containing kanamycins (50mg/mL) is coated on, is passed through
PCR checking screening positive transformant E.coli BL21 (DE3)/pET-28a (+)-pld, are seeded to containing kanamycins (50mg/
ML overnight incubation in LB fluid nutrient mediums).It is new that next day is inoculated in the 50mL containing kanamycins (50mg/mL) by 1% inoculum concentration
In fresh LB fluid nutrient mediums, 37 DEG C of culture about 3h add IPTG and induced, make its final concentration of when OD600 is 1.0
After 0.5mM, 25 DEG C of induction 12h, recombinant bacterium shows phospholipase D activity.
Embodiment 5
This example demonstrates that the Western Blot analysis processes of phospholipase D.
By the restructuring fermented liquid of above-mentioned acquisition at 4 DEG C, 20min is centrifuged under the conditions of 8,000rpm, removes supernatant and collects bacterium
Body is precipitated, and thalline goes the removal of impurity one time with 5ml Tris-HCl (pH7.5) buffer solution for cleaning, and 8,000rpm centrifuge 10min, again
Remove supernatant and collect bacterial sediment, suspended with 5mL Tris-HCl (pH7.5) buffer solution, be prepared into bacteria suspension.Bacteria suspension is existed
It is placed in ice-water bath and carries out ultrasonication using cell Ultrasonic Cell Disruptor, condition is：Work 200 and circulate under 200W power, often
Individual circulation work 4s stops 6s.At 4 DEG C after crushing completely, 20min is centrifuged under the conditions of 12,000rpm, gained supernatant is crude enzyme liquid.
Pass through BCA protein quantification kit measurement protein contents.After preparative separation glue and concentration glue, 80 μ L samples are taken to add 20 μ L
5 x loading buffer, fully mix makes albuminous degeneration after placement 5min in boiling water bath.According to the concentration for determining albumen
Loading is carried out, and adds Marker.Upper strata concentrates glue 80V voltages, when sample to separation gel (about 30min), with 100V electricity
Pressure, closes power supply at sample to separation gel bottom about 1cm, and electrophoresis terminates.It is clean with distilled water flushing after end, make in electric current
Under, protein is set to be transferred to from glue on solid phase carrier PVDF (PVDF membrane).Transfer is closed after finishing, and is incubated
Primary antibody and secondary antibody, finally carry out colour developing and take pictures to confirm the expression of destination protein, as a result show that phospholipase D being capable of clear table
Reach, and molecular size range is about 60kDa (Fig. 2).
Embodiment 6
This example demonstrates that the catalytic characteristics of phospholipase D.
(1) respectively in 40mM pH 4.0-6.0 sodium acetate buffer, pH 6.0-8.0 phosphate buffer, pH
(unit of pH intervals 0.5) is reacted in 8.0-10 Tris-HCl buffer solutions, determines the enzyme activity under different pH, the enzyme
Optimal reaction pH is 7.5, and the 75%~100% of highest enzyme activity can be reached in the scope of pH6.5~8.5.Separately take fresh enzyme liquid point
60min is not placed in above-mentioned buffer solution, is pulled back to after pH 8.0, remaining enzyme activity is determined in the way of standard test enzyme activity,
Restructuring phospholipase D table under conditions of pH6.5~8.5 is relatively stablized, and can keep 70% or so relative enzyme activity (Fig. 3).
(2) appropriate enzyme liquid is taken, temperature spot (5 DEG C of interval) is selected in the range of 20 DEG C to 80 DEG C respectively, determines it in difference
At a temperature of enzyme activity, the optimal reactive temperature of the enzyme is 60 DEG C.Fresh enzyme liquid is separately taken to be incubated respectively in above-mentioned different temperatures
Cooled down immediately after 60min, remaining enzyme activity is determined in the way of standard test enzyme activity, enzyme enzyme activity phase under the conditions of 20~55 DEG C
To stable, more than 65% relative enzyme activity (Fig. 4) can be maintained.
(3) KCl, MgCl are taken2、BaCl2、CaCl2、CoCl2、NaCl、CuCl2、MnCl2、LiCl、FeCl3、FeCl2、
AgNO3、ZnSO4、Al2(SO4)3Etc. 50mM mother liquors are configured to, the mother liquor prepared is separately added into the enzyme liquid suitably diluted, eventually
Concentration is 5mM.After 37 DEG C of insulation 30min, remnant enzyme activity is determined according to enzyme activity determination method, as a result as shown in table 1, with being not added with
The blank control group of any metal ion is compared, Na+、Mn2+And Mg2+Do not show to be obviously promoted effect；Ba2+, Co2+With
Ca2+There are obvious facilitation, wherein Ca to enzyme activity2+Facilitation is most obvious, and 134.1% is reached with respect to enzyme activity；In addition, K+、Cu2+、Li+、Fe3+、Al3+And Zn2+There is less facilitation；And Fe2+And Ag+There is obvious suppression to phospholipase D enzyme activity
Make and use.
Influence of the metal ion of table 1 to enzyme activity
| Reagent | With respect to enzyme activity (%) | Reagent | With respect to enzyme activity (%) |
| Control group | 100±0.6 | Mn2+ | 101.7±1.5 |
| K+ | 105.2±1.5 | Li+ | 107.6±0.8 |
| Mg2+ | 101.1±0.3 | Fe3+ | 105.9±2.1 |
| Ba2+ | 118.2±1.5 | Fe2+ | 56.0±2.2 |
| Ca2+ | 134.1±0.2 | Ag+ | 27.0±1.2 |
| Co2+ | 117.8±1.8 | Zn2+ | 109.7±0.6 |
| Na+ | 101.5±1.2 | Al3+ | 109.3±2.4 |
| Cu2+ | 108.2±0.6 |
(4) protein inhibitor is taken：Beta -mercaptoethanol, DTT, PMSF, EDTA, SDS etc. are configured to 50mM mother liquors, appropriate dilute
The mother liquor prepared, final concentration of 5mM are separately added into the enzyme liquid released.After 37 DEG C of insulation 30min, determined according to enzyme activity determination method
Remnant enzyme activity, as a result as shown in table 2,2-ME, DTT, SDS and PMSF have certain inhibitory action to phospholipase D, and EDTA pairs
The enzyme shows stronger inhibitory action.
Influence of the protein inhibitor of table 2 to enzyme activity
| Reagent | Concentration (mM) | With respect to enzyme activity (%) |
| None | 0 | 100±0.3 |
| 2-ME | 5 | 75±0.9 |
| DTT | 5 | 80±1.1 |
| SDS | 5 | 50±2.1 |
| PMSF | 5 | 40±0.3 |
| EDTA | 5 | 6±0.6 |
Embodiment 7
This example demonstrates that restructuring phospholipase D carries out phosphatidylserine synthetic test.
Weigh 80mg PC60 to be dissolved in 8mL ethyl acetate, weigh 120mg Serines and 6.66mg CaCl2Dissolving
In 4mL phospholipase D crude enzyme liquids, organic phase and aqueous phase are sufficiently mixed uniformly after 40 DEG C, 10h is reacted under the conditions of 180rpm.
As a result as shown in figure 5, under restructuring phospholipase D catalytic action, in 14.2min it can be seen that there is phosphatidylserine generation, passing through
The yield for calculating phosphatidylserine is 0.2g/L, and the life for having phosphatidylserine is not seen in blank control group
Into, illustrate the ability that the restructuring phospholipase D has catalysis substrate phosphatidyl choline and Serine synthetic phospholipid acyl serine,
Food, medicine and healthcare industry have larger application prospect, and recombinant bacterium fermentation period is short, are suitable for industrialization large-scale
Production.
It is of the invention by the scope described by soverlay technique scheme, Yi Jiquan the invention is not restricted to these disclosed embodiments
The various modifications and equivalence changes of sharp claimed range, on the premise of without departing from the technical solution of the present invention, to the present invention
Any modification or improvement that the those skilled in the art made easily realize belong to scope of the present invention.
SEQ ID NO：1
SEQ DNAMAN1: 1614 bp;
Composition 347 A; 435 C; 484 G; 348 T; 0 OTHER
Percentage: 21.5% A; 27.0% C; 30.0% G; 21.6% T; 0.0%OTHER
Molecular Weight (kDa): ssDNA: 499.35 dsDNA: 995.14
ORIGIN
1 CTGCTGCGTC GCCTGCATCG TCTGACCCGT CGCGCAACGG TGTCCGCAGT TCTGCTGGCA
61 CTGCTGCCGG CAGGTCCGGC TCTGGCAGCA GGTGACGCAC CGGGTGCCCC GCACCTGGAT
121 GCGGTTGAAC GTACCCTGCG TCAGGTCTCA CCGGGTCTGG AAGGTGACGT TTGGCAACGT
181 ACGGAGGGTA ACAAACTGGA TGCCAGCGCA GCTGACCCGT CTGATTGGCT GCTGCAGACC
241 CCGGGTTGCT GGGGTGATGC ACATTGTGCT GAACGTCCGG GCACCAAACG CCTGCTGGCT
301 AAAATGACGG AAAATATTTC TCGCGCACGT CGCACCGTGG ATATCAGTAC GCTGGCGCCG
361 TTTCCGGACG GTGCTTTCCA GGATGCGATT GCGGCCGGCC TGAAAGCGAG TGTTGCCTCC
421 GGTAACAAAC CGCGTGTGCG TGTTCTGGTC GGCGCAGCTC CGCTGTATCA CCTGAATGTG
481 CGTCCGAGCG CCTACCTGGA TGACCTGCGT GCACGTCTGG GTACCGCAGC AGATGCAGTT
541 ACGCTGAACG TCGCATCTAT GACCACGAGT AAAACCGCGT TTTCCTGGAA TCATTCAAAA
601 CTGCTGGTTG TGGACGGCGA AAGCGCCATT ACGGGCGGTA TCAACGATTG GAAAAATGAC
661 TATGTTGATA CCGCACACCC GGTCACGGAT GTGGACCTGG CACTGACCGG TCCGGCAGCT
721 TCGACGGCAG GTCGCTATCT GGATACCCTG TGGGGCTGGA CGTGCGGCAA CAAAGGTAAT
781 ATCGCGAGCG TGTGGTACGC AGCATCGGCA GGTGCAAGCT GTATGGCCGA TCTGGAAAAA
841 GAAGCCAACC CGAAACCGGC AGGTCCGACC GGTAATGTGC CGGTTATTGC GGTGGGCGGT
901 CTGGGCGTTG GTATCAAAGA TAAAGACGCA AGCTCTGCTT ATCGTCCGGA ACTGCCGTCT
961 GCACCGGACA CCAAATGCGT CGTGGGCCTG CATGATAACA CCAATGCCGA TCGTGACTAC
1021 GATACGGTGA ACCCGGAAGA ATCAGCACTG CGTGCCCTGG TGGGTTCGGC AGAAAAACAT
1081 GTTGAAATTA GCCAGCAAGA TCTGCACGCT ACCTGTCCGC CGATCGCGAA ATATGACGTT
1141 CGCCTGTACG ATACGCTGGC GAAGAAAATT GCATCCGGTG TCAAAGTGCG TATCGTTGTC
1201 TCAGATCCGG CAAACCGTGG TACCGTGGGT TCTGGCGGTT ATAGTCAGAT TAAATCCCTG
1261 ACGGAAATCT CTGACGTCCT GCGTAATCGT CTGGCACTGG TGACCGGCGG TCAGGATCAA
1321 GCAAAAAACG CTCTGTGCAG TAATGTGCAG CTGGCTTCAT TCCGCTCGGC AAAAAGCCCG
1381 ACCTGGGCTG ACGGTCACGC ATATCCGCTG CATCACAAAC TGGTTTCAGT CGATGGCTCG
1441 GCGTTTTATG TTGGTAGTAA AAATCTGTAC CCGTCCTGGC TGCAGGACTT CGGCTACATT
1501 GTCGAAGATC GTACCGCAGC TGGTCAACTG GATGCCAAAC TGCTGGCACC GGAATGGGAA
1561 TATGCTCAGG ACACCGCGAT CGTCGATTAC CAACGCGGTG TGTGTAGTCT GTAA
SEQ ID NO：2
Universal code
Total amino acid number:538
1 MLRRLHRLTR RATVSAVLLA LLPAGPALAA GDAPGAPHLD AVERTLRQVS PGLEGDVWQR
61 TEGNKLDASA ADPSDWLLQT PGCWGDAHCA ERPGTKRLLA KMTENISRAR RTVDISTLAP
121 FPDGAFQDAI AAGLKASVAS GNKPRVRVLV GAAPLYHLNV RPSAYLDDLR ARLGTAADAV
181 TLNVASMTTS KTAFSWNHSK LLVVDGESAI TGGINDWKND YVDTAHPVTD VDLALTGPAA
241 STAGRYLDTL WGWTCGNKGN IASVWYAASA GASCMADLEK EANPKPAGPT GNVPVIAVGG
301 LGVGIKDKDA SSAYRPELPS APDTKCVVGL HDNTNADRDY DTVNPEESAL RALVGSAEKH
361 VEISQQDLHA TCPPIAKYDV RLYDTLAKKI ASGVKVRIVV SDPANRGTVG SGGYSQIKSL
421 TEISDVLRNR LALVTGGQDQ AKNALCSNVQ LASFRSAKSP TWADGHAYPL HHKLVSVDGS
481 AFYVGSKNLY PSWLQDFGYI VEDRTAAGQL DAKLLAPEWE YAQDTAIVDY QRGVCSL
Claims (4)
1. a kind of plasmid for being used to carry phospholipase D encoding gene, it is characterised in that：PET series, pMA5, pWB may be selected in plasmid
Any of series, pPICZ α, PIC9K, pDXW series, and contain SEQ ID NO：Nucleotide sequence shown in 1.
2. a kind of be used to carry the plasmid of phospholipase D encoding gene, it is characterised in that entrained phospholipase D encoding gene with
SEQ ID NO：Nucleotide sequence shown in 1 has the homology of 90% and the above.
3. a kind of production method for being used to produce the recombinant bacterial strain of phospholipase D, it is characterised in that：By the matter described in claim 1
Grain is imported in foreign host, and described foreign host may be selected from Escherichia coli Escherichia, bacillus, rod
Any of bacillus Corynebacterium, yeast Yeasts, filamentous fungi filamentous fungi.
4. a kind of phospholipase D is applied to the method that phosphatidylserine is synthesized, it is characterised in that：By the weight described in claim 3
Group bacterium is produced in the biphasic reaction system for the organic-aqueous that phospholipase D adds phosphatidyl choline and Serine, in 20-60
Oscillating reactions 5-40h at a temperature of DEG C, has successfully synthesized, can be applied to medicine, food through HPLC detection product phosphatidylserines
Product and healthcare field.
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Cited By (5)
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| CN109321538A (en) * | 2018-09-07 | 2019-02-12 | 江南大学 | A kind of leucine dehydrogenase obtained based on database gene excavating method |
| CN109486790A (en) * | 2018-12-10 | 2019-03-19 | 南通励成生物工程有限公司 | A method of phosphatidyl serine is prepared with phospholipase D conversion |
| CN110004124A (en) * | 2019-02-28 | 2019-07-12 | 江南大学 | A kind of encoding gene of phospholipase D and its expression and application |
| CN112424350A (en) * | 2018-07-30 | 2021-02-26 | 美天施生物科技有限两合公司 | Non-specific nucleases from pseudomonas |
| CN112899256A (en) * | 2021-01-29 | 2021-06-04 | 华南理工大学 | Low-temperature-resistant phospholipase D from Antarctic bacteria and preparation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112424350A (en) * | 2018-07-30 | 2021-02-26 | 美天施生物科技有限两合公司 | Non-specific nucleases from pseudomonas |
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| CN110004124A (en) * | 2019-02-28 | 2019-07-12 | 江南大学 | A kind of encoding gene of phospholipase D and its expression and application |
| CN112899256A (en) * | 2021-01-29 | 2021-06-04 | 华南理工大学 | Low-temperature-resistant phospholipase D from Antarctic bacteria and preparation method and application thereof |
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