CN103509748B - A kind ofly produce the recombination bacillus coli of Phospholipase C and prepare the methods and applications of Phospholipase C - Google Patents
A kind ofly produce the recombination bacillus coli of Phospholipase C and prepare the methods and applications of Phospholipase C Download PDFInfo
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Abstract
The invention discloses and a kind ofly produce the recombination bacillus coli of Phospholipase C and prepare the methods and applications of Phospholipase C, the steps include: the preparation of A, phospholipase C gene: the design of PCR primer and the amplification of phospholipase C gene fragment.The structure of B, recombinant plasmid: through EcoR? I and Not? I enzyme is cut size is the phospholipase C gene fragment of 1158bp, is cloned on high copy number plasmid pET28a, obtains recombinant plasmid pET28a-P.f-PLC; The structure of C, engineering bacteria: plasmid pET28a-P.f-PLC is imported to E.coil with method for transformation? BL21 (DE3).Liquid fermenting can produce great-hearted Phospholipase C, and inside and outside born of the same parents, enzyme activity can reach 408.5U/ml.In oil and fat refining, the field such as phospholipid modified, medical, there is certain application prospect.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of intestinal bacteria producing Phospholipase C, also relate to simultaneously and a kind ofly produce the application in Vegetable oil lipoprotein comes unstuck etc. of the colibacillary preparation method of Phospholipase C and the Phospholipase C of this project bacterium fermentative production.
Background technology
Phospholipase C (PLC,: EC3.1.4.3) be the lytic enzyme of single-minded hydrolysis natural phospholipid Sn-3 position phosphatidyl ester bond, second messenger is played a part in the vital movement of biology, be distributed widely in bacterium, yeast, Acarasiales, fruit bat, the frog and various mammalian body, can be applicable to medicine, feed improvement and field of food industry.Medicine aspect, for developing medicament for resisting platelet aggregation, antithrombotic and cardiovascular and cerebrovascular diseases.In feed improvement, Phospholipid hydrolase makes an addition to hydrolytic phosphatide in feed, improves the utilising efficiency of feed and promotes growth of animal.In foodstuffs industry, PLC can be applied to fat degumming; Simultaneously because Phospholipid hydrolase can make dough form colloidal complex, reduce starch retrogradation, be applied to and cure industry.
In recent years, along with PLC is in the widespread use in the fields such as food, medicine, feed, Chinese scholars has carried out large quantifier elimination to it.Compared with the PLC originated with other, microorganism has that growth and breeding is fast, culture cycle is short, yield of enzyme high.
Though the domestic research to Phospholipase C is started late, but also achieve certain achievement, such as: the people such as Chen Taos in 2005 carry out three ultraviolets and the process of twice Co60-gamma-ray and mutagenesis to the product PLC bacterial strain BacilluscereusShenzhen754-1 screened, filter out the mutagenic fungi of three plant height PLC activity, enzyme live reach 14.878 respectively, 16.450,16.400U/ml (Chen Tao, Wang Changgao, Liu Xiaohui, what is conquered east. the mutagenic and breeding of high yield Phospholipase C bacterial strain. and research and development of natural products, 2005,17 (6): 712-716.); Within 2007, high woods carries out the optimization of culture condition to BacilluscereusShenzhen754-1, enzyme is lived be increased to 26U/mL (high woods. the optimizing research of the screening of Phospholipase C superior strain, qualification and culture condition. the master thesis of Agricultural University Of Anhui, 2007); Zhan Yi in 2010 relaxes and screens a strain BacilluscereusZ-13, the precipitation circle (milky white precipitate circle) of the PLC that this bacterial strain produces is measured with egg yolk agar cylinder plate method, its diameter can reach 30mm, enzyme live reach 23.31U/ml (Zhan Yishu. produce screening and the zymology Quality Research thereof of Phospholipase C bacterial strain. the master thesis of Agricultural University Of Hunan, 2010).
Compared with the PLC of plant and animal material, the PLC in mentioned microorganism source has the shorter production cycle, but still there is following drawback: as wild strain separation and purification cost is large, great majority have potential pathogenicity bo, there is certain hidden danger in food safety; Engineering strain its build and produce enzymatic process comparatively complicated, yield of enzyme need improve.
In vegetable oil and fat refining, need phosphatide removing (namely coming unstuck) in crude oil, if do not come unstuck, grease will turn black when being heated to certain temperature and produce black smoke, traditional degumming technology is the method adopting alkalization to learn, and namely in crude oil, adds NaOH, phospholipid conversion is become sodium soap, by centrifugal removing, this method oil loss and energy consumption comparatively large and the waste water produced and Chinese honey locust contaminate environment.And Phospholipase C is applied to vegetable oil and fat refining, Phospholipase C is utilized to become to be easy to the phosphoric acid ester be dissolved in water and the triglyceride be dissolved in oil by the phospholipid conversion in grease, except the phosphoric acid ester in degrease, the object of coming unstuck can be reached, do not produce containing alkali waste water and Chinese honey locust through centrifugal again.Study on enzymatic degumming has become the development trend of oil refining process with its good economic benefit, remarkable environment-friendly advantage and simple production technique.
Enzymatic degumming technique also obtains applying more and more widely along with deepening continuously of Phospholipid hydrolase preparation research, and Study on enzymatic degumming carries out industrial applications at Oils and fats enterprises such as Bungay company of the U.S., German Bungay mannheim companies in succession.South Sea oil prodution industry company limited is the domestic first enterprise carrying out the research of enzymatic degumming process pilot.South China Science & Engineering University and Novozymes Company cooperate, and carry out great many of experiments with enzymatic degumming, and result proves that the crude oil of enzymatic degumming to different mass has great adaptability, all applicable to soybean oil, rapeseed oil, sunflower seed oil, Semen Maydis oil, rice bran wet goods.
Summary of the invention
In view of Problems existing in above-mentioned and/or existing enzyme genetically engineered and enzyme engineering field, propose the present invention.
For the problems referred to above that prior art exists, a strain is the object of the present invention is to provide to produce the recombination bacillus coli of Phospholipase C and prepare the method for Phospholipase C with it.Utilize this bacterial strain to produce Phospholipase C by liquid fermenting, yield of enzyme and enzymic activity high, security is good, and enzymatic process processed is simple, and cost is low.
A further object of the invention there are provided a kind of application of intestinal bacteria in Vegetable oil lipoprotein (soybean oil, rapeseed oil, rice bran wet goods) comes unstuck of producing Phospholipase C.The Phospholipase C utilizing Escherichia coli fermentation to produce and Yelkin TTS (PC) and phosphatidylethanolamine (PE) react, and generate triglyceride and water-soluble phosphate, thus reduce the emulsification situation in final operation.Can colloid be separated cleaner in centrifugal separation processes like this, decrease the loss of neutral oil simultaneously.In addition, the triglyceride (DG) generated in Phospholipase C scouring processes is also a kind of serendipitous oily product, exists all the time in whole refinery processes.Water-soluble phosphate is easy to removing through hydration degum, makes the phosphorus content≤10ppm in grease (being equivalent to phospholipids content lower than 0.26 ‰).Compared with existing chemical method, there is environmental protection, the advantage that energy-conservation, oil yield is high.
Technical scheme of the present invention is as follows:
A kind of recombination bacillus coli (Eschierichiacoil) BL21 (DE3)-pET-P.f-PLC producing Phospholipase C, deposit number is CCTCCNo.M2013299.Recombination bacillus coli EschierichiacoilBL21 (DE3)-pET-P.f-PLC, the CCTCCNo.M2013299 of described product Phospholipase C, obtained by following method:
(1) take deposit number as the genome of Pseudomonas fluorescens (Pseudomonasfluorescens) of CCTCCNo.M2013298 be template, clone obtains phospholipase C gene, and its base sequence is as shown in SEQIDNO:1;
(2) step (1) gained phospholipase C gene is cloned into expression vector pET-28a(+) on, obtain recombinant vectors;
(3) by step (2) gained recombinant vectors transformation of E. coli competent cell, build and obtain recombination bacillus coli.
Present invention also offers a kind of recombination bacillus coli (Eschierichiacoil) BL21 (DE3)-pET-P.f-PLC of above-mentioned product Phospholipase C, CCTCCNo.M2013299 prepares the method for Phospholipase C, be carry out liquid fermenting with this recombination bacillus coli for fermentation strain, prepare Phospholipase C.
Its concrete steps are as follows:
(1) seed culture: the recombination bacillus coli BL21(DE3 by described product Phospholipase C)-pET-P.f-PLC, CCTCCNo.M2013299 be inoculated in seed culture medium, in 30 ~ 37 DEG C of shaking culture 8 ~ 12h, shaking speed 150 ~ 200r/min;
(2) liquid fermentation and culture: the inoculum size of cultivating gained activated seed liquid by volume per-cent 3 ~ 10% through step (1) is seeded to fermention medium, in 30 ~ 37 DEG C of shaking culture 4 ~ 6h hour, shaking speed 150 ~ 200r/min; Add lactose again, in 20 ~ 30 DEG C of vibration inducing culture 14 ~ 22h, shaking speed 150 ~ 200r/min; Finally add TritonX-100, under the same terms, continue vibration inducing culture 18 ~ 36h, ferment complete, obtain fermented liquid;
(3) extraction of crude enzyme liquid: by centrifugal for step (2) gained fermented liquid; Collect supernatant liquor, be the outer crude enzyme liquid of born of the same parents; Collect somatic cells precipitation and ultrasonication, by centrifugal for gained ultrasonication liquid, get supernatant, be crude enzyme liquid in born of the same parents.
Its further technical scheme is:
The described seed culture based component of step (1) is counted by grams per liter: peptone 9 ~ 11g, yeast powder 4 ~ 6g, NaCl9 ~ 11g, and all the other compositions are water.
Step (2) described fermentation medium components is counted by grams per liter: peptone 9 ~ 11g, yeast powder 5 ~ 24g, glycerine 0 ~ 5g, and all the other compositions are 0.25mol/LTris-HCl (pH7.2).
The addition of step (2) described lactose is often liter of described fermention medium 0 ~ 5g.
The addition of step (2) described TritonX-100 is often liter of described fermention medium 0 ~ 10g.
Intestinal bacteria provided by the present invention (Escherichiacoli) BL21 (DE3)-pET-P.f-PLC, be preserved in China typical culture collection center (CCTCC) on June 28th, 2013, deposit number: CCTCCNo.M2013299, address: China, Wuhan, Wuhan University.This bacterial strain is hereinafter referred to as intestinal bacteria EscherichiacoliBL21 (DE3)-pET-P.f-PLC, CCTCCNo.M2013299.
The technique effect that the present invention is useful is:
Compared with the wild isolated strains of existing product Phospholipase C and engineering strain, the present invention constructs a strain goal gene and derives from Pseudomonas fluorescens (Pseudomonasfluorescens), can genetically engineered recombinant escherichia coli EschierichiacoilBL21 (the DE3)-pET-P.f-PLC of high yield Phospholipase C, CCTCCNo.M2013299, this bacterial strain is utilized to produce Phospholipase C by liquid fermenting, yield of enzyme is high, its enzymic activity reaches as high as 408.5U/ml, and security is good; This enzyme method production technique processed is simple, cost is low, be easy to technique amplifies, and has certain application prospect in oil and fat refining, the field such as phospholipid modified, medical.
The measuring method that Phospholipase C enzyme is lived is as follows:
(1) borax egg plate method mensuration enzyme is lived.Fermented liquid is carried out ultrasonication, the supernatant liquor got after 100 ~ 200 μ L fragmentations adds in the Oxford cup be equidistantly placed on borax egg yolk agar flat board, in 37 ° of C incubation 12 ~ 24h, represent enzyme height alive respectively with the size of the decomposition circle generated (transparent circle or muddy circle).
(2) p-NPPC standard measure mensuration enzyme is lived.P-NPPC is a kind of substrate structure analogue of Yelkin TTS, Phospholipase C hydrolyzable p-NPPC generates p-NP, p-NP is a kind of yellow substance, maximum absorption band is had at 410nm place, utilize spectrophotometry fermented liquid at the light absorption value at 410nm place, can reflect that Phospholipase C hydrolysis p-NPPC produces the amount of p-NP, quantitative Analysis can go out corresponding enzyme activity size according to p-NP typical curve; In 1L reaction solution system, it is composed as follows: 10mmolp-NPPC, 0.25mol/LTris-HCl (pH7.2), adds the fermented liquid of 100 μ L in 1mL reaction system, reacts 30min in 37 ° of C; Enzyme activity unit is defined as follows: at pH7.2, and temperature is under the condition of 37 ° of C, and the amount of the enzyme that per minute hydrolysis p-NPPC produces needed for 1nmol p-NP is 1 enzyme activity unit (U).
In said determination method, described borax egg yolk medium composition is as follows: NaCl6.6g/L, boric acid 10.9g/L, borax 1.9g/L, agar 10g/L, yolk liquid 10g/L, and all the other compositions are water, and pH7.2 ~ 7.4, in 1 × 10
5pa autoclaving 20min.Shake up rear pour plate, to obtain final product.
Produce the application of engineering bacteria in removing raw plant oil (soybean oil, rapeseed oil, Rice pollard oil) in phosphatide for Phospholipase C, comprise, the preheating of raw plant oil and low-kappa number; Add Phospholipase C as claimed in claim 3 to react; Go out enzyme, and centrifugation completes comes unstuck.
As a kind of preferred version of application of the present invention, wherein: described preheating is for being preheating to 80 DEG C; Described low-kappa number is for adding citric acid homogeneous; Described add Phospholipase C as claimed in claim 3 carry out reaction be under 45 DEG C of conditions react 2 hours; Described centrifugation is get upper oil phase under 5000rpm condition centrifugal 5 minutes.
Get vegetables oil crude oil 100g (phosphorus content 400-600ppm), be preheated to 80 DEG C, add 2.5mol/L citric acid 0.125ml, homogeneous 1min, maintain 30min in this temperature.Be cooled to 40 ~ 45 DEG C, add 4mol/LNaOH solution and 2 ~ 3% distilled water mix, then add the Recombinant E. coli Fermentation Broth (i.e. Phospholipase C enzyme liquid) of 0.5ml, 40 ~ 45 DEG C of reactions 2 ~ 3 hours.Sampling, measures containing phosphorus content.
Adopt and the phosphorus content in vegetables oil can be dropped to 10ppm (being equivalent to phospholipids content lower than 0.26 ‰) in this way and below, meet the requirement of physical refining.
Embodiment
Set forth a lot of detail in the following description so that fully understand the present invention, but the present invention can also adopt other to be different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar popularization when intension of the present invention, therefore the present invention is by the restriction of following public specific embodiment.
In following embodiment use experiment material as follows: competent escherichia coli cell E.coliJM109 and E.coliBL21 (DE3) be laboratory preserve; Pseudomonas fluorescens (Pseudomonasfluorescens) is this laboratory screening, and deposit number is: CCTCCNo.M2013298; PMD18T-simpleVector, T4DNA ligase enzyme and 10 × T4 ligase enzyme Buffer are purchased from precious biotechnology (Dalian) company limited; PET-28a (+) is purchased from Novagen; Restriction enzyme EcoRI, NotI and total Buffer are purchased from NewEnglandBiolabs.
The clone of embodiment 1 phospholipase C gene
According to the primers of the phospholipase C gene (NC_017911.1) of the PseudomonasfluorescensA506 that NCBI provides, wherein,
Upstream primer 5'CG
gAATTCaTGTCAGGTCTTGAACTCGC3', (underscore is EcoRI restriction enzyme site); Downstream primer 5'TT
gCGGCCGCtTAGTTGGCGGGTTGGTTTGGC3', (underscore is NotI restriction enzyme site).
Take deposit number as the genomic dna of the Pseudomonas fluorescens (Pseudomonasfluorescens) of CCTCCNo.M2013298 be template, the method of PCR clone is used to obtain phospholipase C gene, and be connected with carrier pMD18T-simple and build cloning vector pMD-plc, by this cloning vector transformation of E. coli competent cell E.coliJM109, select positive colony, sequence verification, its base sequence is as shown in SEQIDNO:1, and result shows that phospholipase C gene is cloned successfully.Blast sequence alignment analysis shows, and the homology of this gene order and existing phospholipase C gene sequence is lower, is up to 89%, has widened the gene source of Phospholipase C.
The structure of embodiment 2 plasmid pET-P.f-PLC
Carry out double digestion with EcoRI and NotI to recombinant vectors pMD-plc and expression vector pET-28a (+), endonuclease reaction system is that each 0.5 μ L, EcoRI and the NotI of 10 μ L:pET-28a (+) μ L, EcoRI and NotI shares Buffer1 μ L.Reclaim goal gene and carrier DNA, and connect with T4DNA ligase enzyme, ligation system 10 μ L: target gene 5 μ L, carrier DNA 3 μ L, 10 × T4 ligase enzyme Buffer1 μ L, T4DNA ligase enzyme 1 μ L, in 16 DEG C of reaction 12h.
To connect product conversion competent escherichia coli cell E.coliJM109, method for transformation is as follows:
(1) get E.coliJM109100 μ L and be placed in aseptic 1.5mlEP pipe;
(2) add above-mentioned connection product and mix gently;
(3) above-mentioned EP pipe is placed in 42 DEG C of waters bath with thermostatic control, timing 1.5min, does not shake EP pipe;
(4) EP pipe is shifted in ice bath, cooling 2min;
(5) often pipe adds aseptic SOB substratum 800 μ L, is placed in 37 DEG C of incubator recovery 1h;
(6) with the centrifugal 2min of the rotating speed of 8000r/min, suck 800 μ L supernatant substratum, with liquid-transfering gun pressure-vaccum residue substratum and cell gently, and be transferred to the LB solid plate containing final concentration 100 μ g/ml kantlex, smoothen and cultivate 16 ~ 24h in 37 DEG C of incubators;
(7) picking positive colony, and verify through EcoRI and NotI double digestion.
The structure of embodiment 3 recombination bacillus coli EschierichiacoilBL21 (DE3)-pET-P.f-PLC, CCTCCNo.M2013299
By recombinant plasmid pET-plc transformation of E. coli BL21 (DE3) built, method for transformation is as follows:
(1) the EP pipe that E.coliBL21 (DE3) 100 μ L is placed in aseptic 1.5ml is got;
(2) add above-mentioned recombinant plasmid pET-plc and mix gently;
(3) above-mentioned EP pipe is placed in 42 DEG C of waters bath with thermostatic control, accurate timing 1.5min, does not shake EP pipe;
(4) EP pipe is shifted in ice bath, cooling 2min;
(5) often pipe adds sterile LB medium 800 μ L, is placed in 37 DEG C of incubator recovery 1h;
(6) with the centrifugal 2min of the rotating speed of 8000r/min, suck 800 μ L supernatant substratum, with liquid-transfering gun pressure-vaccum residue substratum and cell gently, and be transferred to the LB solid plate of final concentration 100 μ g/ml kantlex, smoothen and cultivate 16 ~ 24 hours in 37 DEG C of incubators;
(7) picking transformant, digestion verification recombination bacillus coli BL21 (DE3)-pET-plc successfully constructs, and the glycerine being 30% with isopyknic volume fraction mixes, in-70 DEG C of preservations.
Phospholipase C is prepared with recombination bacillus coli EschierichiacoilBL21 (DE3)-pET-P.f-PLC, CCTCCNo.M2013299
Embodiment 4
(1) seed culture: recombination bacillus coli EschierichiacoilBL21 (the DE3)-pET-P.f-PLC getting the above-mentioned preservation of 50 μ L, CCTCCNo.M2013299 is inoculated in seed culture medium, in 37 DEG C of shaking culture 10h, shaking speed 150r/min on Clothoid type shaking table; Above-mentioned seed culture based component is counted by grams per liter: peptone 10g, yeast powder 5g, NaCl10g, and all the other compositions are water, sterilizing 20min under 1 × 105Pa high pressure.
(2) liquid fermentation and culture: the inoculum size of cultivating gained activated seed liquid by volume per-cent 5% through step (1) is seeded to fermention medium, fermention medium is loaded on 250ml triangular flask, liquid amount is 50ml, in 37 DEG C of shaking culture 4h hour, shaking speed 200r/min on Clothoid type shaking table; Adding lactose to final concentration is 0.1g/L, and in 18 DEG C of vibration inducing culture 30h on Clothoid type shaking table, shaking speed is 150r/min; Ferment complete, obtain fermented liquid; Above-mentioned fermentation medium components is counted by grams per liter: peptone 10g, yeast powder 5g, and all the other compositions are 0.25mol/LTris-HCl (pH7.2), sterilizing 20min under 1 × 105Pa high pressure.
(3) extraction of crude enzyme liquid: by step (2) gained fermented liquid in 4 DEG C of centrifugal 10min, rotating speed 8000r/min; Collection somatic cells precipitates, and with 50mmol/LTris-HCl (pH7.2) resuspended thalline, is placed in Ultrasonic Cell Disruptor ultrasonication 10min, by centrifugal for gained ultrasonication liquid, gets supernatant, be crude enzyme liquid in born of the same parents.The enzyme utilizing p-NPPC standard measure to record crude enzyme liquid in born of the same parents is lived and is reached 360U/ml.
Embodiment 5
(1) seed culture: recombination bacillus coli EschierichiacoilBL21 (the DE3)-pET-P.f-PLC getting the above-mentioned preservation of 50 μ L, CCTCCNo.M2013299 is inoculated in seed culture medium, in 37 DEG C of shaking culture 10h, shaking speed 150r/min on Clothoid type shaking table; Above-mentioned seed culture based component is counted by grams per liter: peptone 10g, yeast powder 5g, NaCl10g, and all the other compositions are water, sterilizing 20min under 1 × 105Pa high pressure.
(2) liquid fermentation and culture: the inoculum size of cultivating gained activated seed liquid by volume per-cent 5% through step (1) is seeded to fermention medium, fermention medium is loaded on 250ml triangular flask, liquid amount is 50ml, in 37 DEG C of shaking culture 4h hour, shaking speed 200r/min on Clothoid type shaking table; Adding lactose to final concentration is 0.1g/L, and in 18 DEG C of vibration inducing culture 16h on Clothoid type shaking table, shaking speed is 150r/min; ; Adding TritonX-100 again to final concentration is 5g/L, and it is complete that similarity condition continues fermentation 20h fermentation, obtains fermented liquid; Above-mentioned fermentation medium components is counted by grams per liter: peptone 10g, yeast powder 5g, and all the other compositions are 0.25mol/LTris-HCl(pH7.2), sterilizing 20min under 1 × 105Pa high pressure.
(3) extraction of crude enzyme liquid: by step (2) gained fermented liquid in 4 DEG C of centrifugal 10min, rotating speed 8000r/min; Collection somatic cells precipitates, and with 50mmol/LTris-HCl (pH7.2) resuspended thalline, is placed in Ultrasonic Cell Disruptor ultrasonication 10min, by centrifugal for gained ultrasonication liquid, gets supernatant, be crude enzyme liquid in born of the same parents.The enzyme utilizing p-NPPC standard measure to record crude enzyme liquid in born of the same parents is lived and is reached 408.5U/ml.
Embodiment 6 one kinds of Phospholipase C application in degumming of rape oil, the steps include:
A, get 100g crude rapeseed oil (phosphorus content is 186ppm), be preheating to 80 DEG C;
B, the 2.5mol/L citric acid 0.125ml added, after homogeneous 1min, insulation 30min;
C, add 5mol/LNaOH0.25mL, distilled water 3mL, Phospholipase C enzyme liquid (e. coli bl21-pET-P.f-PLC) fermentation obtains, and vigor is 360U/mL) 500 μ L, homogeneous 1min under 2000rpm, react 1 hour under 45 DEG C of conditions.
Centrifugal 5 minutes of D, 5000rpm, get upper oil phase, phosphorus content after adopting GB5537-2008 " Vegetable oil lipoprotein inspection phospholipid determination method " to measure degumming process in oil product is 5.449ppm, reduces about 97.07%, can meet the requirement of physical refining process before phosphorus content ratio comes unstuck.
Embodiment 7 one kinds of Phospholipase C application in soybean oil is come unstuck, the steps include:
A, get 100g crude oil of soybean (phosphorus content is 642ppm), be preheating to 80 DEG C;
B, the 2.5mol/L citric acid 0.125ml added, after homogeneous 1min, insulation 30min;
C, add 5mol/LNaOH0.25mL, distilled water 3mL, Phospholipase C enzyme liquid (e. coli bl21-pET-P.f-PLC) fermentation obtains, and vigor is 360U/mL) 500 μ L, homogeneous 1min under 2000rpm, react 1 hour under 45 DEG C of conditions.
Centrifugal 5 minutes of D, 5000rpm, get upper oil phase, phosphorus content after adopting GB5537-2008 " Vegetable oil lipoprotein inspection phospholipid determination method " to measure degumming process in oil product is 11.857ppm, reduces about 98.15%, can meet the requirement of physical refining process before phosphorus content ratio comes unstuck.
It should be noted that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (5)
1. the recombination bacillus coli producing Phospholipase C (
escherichiacoil) BL21 (DE3)-pET-P.f-PLC, deposit number is CCTCCNo.M2013299.
2. prepare the method for Phospholipase C with the recombination bacillus coli producing Phospholipase C described in claim 1, it is characterized in that: carry out liquid fermenting with the recombination bacillus coli of this product Phospholipase C for fermentation strain, prepare Phospholipase C.
3. prepare the method for Phospholipase C according to claim 2, it is characterized in that: concrete steps are as follows:
(1) seed culture: the recombination bacillus coli BL21(DE3 by described product Phospholipase C)-pET-P.f-PLC, be inoculated in seed culture medium, in 30 ~ 37 DEG C of shaking culture 8 ~ 12h, shaking speed 150 ~ 200r/min;
(2) liquid fermentation and culture: the inoculum size of cultivating gained activated seed liquid by volume per-cent 3 ~ 10% through step (1) is seeded to fermention medium, in 30 ~ 37 DEG C of shaking culture 4 ~ 6h hour, shaking speed 150 ~ 200r/min; Add lactose again, in 20 ~ 30 DEG C of vibration inducing culture 14 ~ 22h, shaking speed 150 ~ 200r/min; Finally add TritonX-100, under the same terms, continue vibration inducing culture 18 ~ 36h, ferment complete, obtain fermented liquid;
(3) extraction of crude enzyme liquid: by centrifugal for step (2) gained fermented liquid; Collect supernatant liquor, be the outer crude enzyme liquid of born of the same parents; Collect somatic cells precipitation and ultrasonication, by centrifugal for gained ultrasonication liquid, get supernatant, be crude enzyme liquid in born of the same parents.
4. prepare the method for Phospholipase C according to claim 3, it is characterized in that: the described seed culture based component of step (1) is counted by grams per liter: peptone 9 ~ 11g, yeast powder 4 ~ 6g, NaCl9 ~ 11g, and all the other compositions are water.
5. a raw plant oil method of coming unstuck, is characterized in that: comprise,
The preheating of raw plant oil and low-kappa number, described preheating is for being preheating to 80 DEG C, and described low-kappa number is for adding citric acid homogeneous;
Add Phospholipase C as claimed in claim 2 to react, react 1 hour under 45 DEG C of conditions;
Go out enzyme, and centrifugation completes comes unstuck, and described centrifugation is get upper oil phase under 5000rpm condition centrifugal 5 minutes.
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| CN105734029B (en) * | 2014-12-12 | 2020-09-25 | 丰益(上海)生物技术研发中心有限公司 | Phospholipase antibacterial peptide |
| CN104878033B (en) * | 2015-04-29 | 2018-08-28 | 江南大学 | A kind of method and its application promoting target protein extracellular expression |
| CN106119237B (en) * | 2016-08-05 | 2019-08-13 | 集美大学 | A kind of method of modifying of recombination phospholipase C |
| CN106754601A (en) * | 2016-12-21 | 2017-05-31 | 江南大学 | A kind of application phospholipase C by intracellular protein extracellular expression method |
| CN110283802B (en) * | 2018-03-19 | 2021-04-23 | 中国科学院遗传与发育生物学研究所 | Soybean non-specific phospholipase GmNPC2 and application of coding gene thereof in regulation and control of vegetable oil metabolism |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002486A (en) * | 2010-10-22 | 2011-04-06 | 北京理工大学 | Phospholipase B from pseudomonas fluorescens and production method thereof |
| KR20110099459A (en) * | 2010-03-02 | 2011-09-08 | 의료법인 성광의료재단 | Production methods of plc-z1 protein inducing ca oscillation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20110099459A (en) * | 2010-03-02 | 2011-09-08 | 의료법인 성광의료재단 | Production methods of plc-z1 protein inducing ca oscillation |
| CN102002486A (en) * | 2010-10-22 | 2011-04-06 | 北京理工大学 | Phospholipase B from pseudomonas fluorescens and production method thereof |
Non-Patent Citations (3)
| Title |
|---|
| "植物油脱胶研究进展;高阴榆 等;《食品科学》;20060930;第27卷(第9期);268页倒数第1段-第269页倒数第1段 * |
| 磷脂酶C用于大豆油脱胶的工艺优化;杨娇 等;《中国油脂》;20121231;第37卷(第12期);14-17 * |
| 重组大肠杆菌表达铜绿假单胞菌溶血性磷脂酶C;赵金星 等;《微生物学报》;20130331;第53卷(第3期);摘要、第260页左栏第3段-261页右栏倒数第1段 * |
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