CN103525744A - Recombinant escherichia coli, method for preparing phospholipase C and application - Google Patents
Recombinant escherichia coli, method for preparing phospholipase C and application Download PDFInfo
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Abstract
The invention discloses recombinant escherichia coli, a method for preparing phospholipase C and application. The recombinant escherichia coli BL21(DE-3)-pET-28a-L.m-phospholipase C (PLC) of which the target gene is from listeria monocyogenes (L.monocytogenes) is utilized as a fermentation strain to carry out liquid fermentation, so as to prepare phospholipase C. The method disclosed by the invention is simple in enzyme production process, short in production cycle and low in cost. A glycerophosphate bond on glyceryl phosphatide C3 can be specifically hydrolyzed to produce diglyceride (DAG) by using recombinant phospholipase C; plant crude oil (soybean oil, colleseed oil, rice bran oil and the like) is degummed by phospholipase C.
Description
Technical field
The present invention relates to a kind of production method of the Phospholipase C that derives from listerisa monocytogenes in mjme and in the application of fat degumming industrial aspect, belong to enzyme genetically engineered and enzyme engineering field.
Background technology
Listerisa monocytogenes in mjme (L.monocytogenes), is a kind of important food-borne pathogens, and growth temperature range is-1.5 ℃-45 ℃, and optimum temperuture is 30 ℃-37 ℃.Single increasing listeria spp is that typical resistance to cold bacterium can be at the refrigerating chamber growth and breeding of general refrigerator, and 20 ℃ of low temperature of ﹣ still can partly be survived, and the food refrigerated time of containing Listeria monocytogenes is longer, and its danger is larger; And the stronger temperature capacity of tool, milk pasturisation temp can not be killed.Amphimicrobian, grows in the micro-aerobic environment that contains CO2 of being everlasting; Not high to nutritional requirement, can form microbial film at body surface has resistant function to antimicrobial substance, also can be acidproof, can resist freeze-thaw repeatedly, and resist drying, has salt tolerance.Single listeria spp that increases can be grown in wildlife, domestic animal, birds, insect, sewage, soil and plant, therefore, single listeria spp that increases often can in ox, sheep, poultry, be detected, in milk-product, fruit, vegetables, cooked meat product, sea-food, also often have pollution in various degree.Clinical demonstration, infects listeria spp disease and comprises miscarriage, septicemia, meningitis etc., the similar influenza of symptom, and often with heating gastroenteritis syndrome.
Single listeria spp that increases can produce two kinds of Phospholipase Cs (PLC): phosphatidylinositols Phospholipid hydrolase (phosphatidylinositol phospholipaseC, PI-PLC) and phosphatidylcholine Phospholipase C (phosphatidylcholin phospholipase C, PC-PLC).The former is by plcA genes encoding, and the latter is by plcB genes encoding.PlcA gene with plcB gene is all and single important gene composition that increases the relevant pathogenicity island 1 (LIPI-1) of listeria spp born of the same parents endoparasitism life cycle.
PLC Main Function is Phosphoric acid glycerol esters ester bond on glyceryl phosphatide C3 position, and hydrolysate is triglyceride (DAG) and phosphotidate (phosphorylcholine, phosphorylethanolamine, phosphoserine and phosphoinositide etc.), in pharmaceutical industries, has important application always.And a composition that can jointly become oil with triglyceride level (TAG) because of hydrolysate DAG at present does not need to be removed, and cause the concern of oils industry, more and more as a kind of emerging enzymatic degumming, with Phospholipid hydrolase, in fat degumming technique, used.It is wide that PLC enzymatic degumming has suitability, reaction conditions is gentle, in production, save the consumption of soda acid chemical, produce hardly soap stock and waste water etc., the economic benefit common advantage of general enzymatic degumming such as rise appreciably, but compared to other enzymatic degumming, as phospholipase A (PLA) enzymatic degumming, PLC enzymatic degumming can not only shorten usually time greatly, can also effectively improve crude oil yield, reduce crude oil loss, common every 500ppm phosphorus just can improve approximately 1% oil yield, and this feature seems particularly important in today of provision price continuous rise.
Abroad to coming unstuck, with the research of Phospholipase C, start to walk early.1988, the propositions such as Graille were processed grease with PLC, and phosphatide is changed into DAG, not only TAG composition can not taken away, can also generating portion " oil ", reduce the loss of grease in refining process.Graille etc. also further research and utilization bacillus cereus prepare PLC zymin, but due to the restriction of preparation cost at that time, have ignored the potential commercial value of PLC.2005, S. lattice drew the matico (Piper angustifolium) watt Pichi strain expressing heterologous PLC by Mut+ phenotype, for the treatment of grease, the phosphatide residual quantity in crude oil can be reduced to below 10ppm.But in view of most of PLC belong to phosphatidylcholine specificity Phospholipase C (PC-PLC), Phospholipid hydrolase choline (PC) and phosphatidylethanolamine (PE) are had to higher specificity, and it is lower to phosphatidylinositols (PI) activity, only with PC-PLC, come unstuck and can not remove the phosphorus in crude oil completely, so Gramatikova proposed PLC and PLA to combine to use to carry out fat degumming first in 2005.2006, Nagasaki was chanted son etc. and is utilized Aspergillus tamarii IAM13907 strain and aspergillus oryzae NBRC4190 strain purifying to obtain a kind of new type PLC, and relative molecular weight is about 87kDa, more stable within the scope of 0-80 ℃, is widely used in foods and cosmetics industry.2007 Nian， Verenium companies have released in AOCS meeting
(PLC), become the most ripe the coming unstuck of current technique PLC product.PLC(BD16449) be that Vince Ciofalo utilizes the recombinant expressed a kind of molecular weight of pichia spp SMD1168 to be about the glycosylated proteinase of 34kDa, safe, be applied to vegetable oil degumming.2008, C.L.g. Dai Dun etc., on the Research foundation of Gramatikova, considered the relative merits of PLA and PLC, have invented the mixed preparation of PC-PLC/PI-PLC and PLA1/PLA2, in order to remove the phosphatide in vegetables oil.Research shows, the enzymatic reaction speed of compound formulation is obviously better than independent use, can say that residual phosphorus content is down to below 3ppm, thereby avoid other auxiliary degumming operations, saved a large amount of chemical reagent and water.Danisco also passes through
(LAT) and the compound formulation of PLC colloid is processed, significantly reduced the refining consumption rate of crude oil.
Domestic fat degumming uses the research of PLC still in the starting stage.Institute of viruses, Wuhan Chen Tao team is devoted to the research of microorganism PLC always, Li Caifeng and Zhan Yishu have screened respectively Vibrio harveyi and the bacillus cereus that produces PLC, Liu Feifei utilize intestinal bacteria and pichia spp recombinant expressed bacillus cereus PLC, Zhao Jinxing has utilized Recombinant protein expression Pseudomonas aeruginosa PLC, but its prepared PLC is not all applied to fat degumming.2003, Sun Chun separation and purification from serratia marcescens fermented supernatant fluid obtained a kind of PLC.2006, Meng Qingfei utilized this enzyme to come unstuck to soybean oil, made the percent hydrolysis of phosphatide in crude oil reach 85.7%, and refining yield has improved 3.08%.Yang Jiao is used PLC to come unstuck for crude oil of soybean, and residual phosphorus amount is reduced to 17.8ppm.2012, utilize in palace etc. sodium alginate and chitosan fixedly PLC carry out crude oil of soybean and come unstuck, phosphorus content is down to 3.8ppm.2013, Liu Lu etc. utilized PLC to carry out crude rapeseed oil to come unstuck, phosphorus content is down to 4.3ppm.
Although PLC Study on enzymatic degumming is relatively late in the application starting of oils industry, development in recent years is very rapid.Therefore develop and there is the autonomous property right of China, at a low price, the enzymatic degumming of high-quality, highly versatile has very important realistic meaning with PLC.
Summary of the invention
Problem in view of existing in above-mentioned and/or existing enzyme genetically engineered and enzyme engineering field, has proposed the present invention.
Therefore, one of them object of the present invention is to provide a kind of recombination bacillus coli.
For solving the problems of the technologies described above, according to an aspect of the present invention, the invention provides following technical scheme: a kind of recombination bacillus coli BL21 (DE3)-pET28a-L.m-PLC, deposit number is CCTCC No.M2013301.
As a kind of constructing plan of recombination bacillus coli BL21 of the present invention (DE3)-pET28a-L.m-PLC, wherein: made by following method:
(1) take the genome of the listerisa monocytogenes in mjme (L.monocytogenes) that deposit number is CICC No.21540 is template, and clone obtains phospholipase C gene, and its base sequence is as shown in SEQ ID NO:1;
(2) step (1) gained phospholipase C gene is cloned into expression vector pET-28a(+) upper, obtain recombinant vectors;
(3) step (2) gained recombinant vectors is transformed to competent escherichia coli cell, build and obtain recombination bacillus coli.
Intestinal bacteria provided by the present invention (Escherichia coli) BL21 (DE3)-pET28a-L.m-PLC, on June 28th, 2013, be preserved in Chinese Typical Representative culture collection center (CCTCC), deposit number: CCTCC No.M2013301, address: China, Wuhan, Wuhan University.This bacterial strain is hereinafter to be referred as-pET28a-L.m-PLC intestinal bacteria (Escherichia coli) BL21(DE3).
Another object of the present invention is to provide a kind of recombination bacillus coli BL21(DE3 that utilizes)-pET28a-L.m-PLC carries out as fermentation strain the method that liquid fermenting is prepared Phospholipase C, its enzymatic process processed is simple, with short production cycle, cost is low, at raw plant oil, has certain application prospect aspect coming unstuck.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: comprise,
(1) seed culture: by described recombination bacillus coli BL21(DE3)/pET-lm-plc, CCTCC No.M2013301 is inoculated in seed culture medium, in 28 ℃～37 ℃ shaking culture 6h～14h, shaking speed 150r/min～220r/min;
(2) liquid fermentation and culture: by through step (1), cultivate gained activated seed liquid by volume the inoculum size of per-cent 1～10% be seeded to fermention medium, in 28～37 ℃ of shaking culture 4h～6h hour, shaking speed 150～220r/min; Add again lactose, in 18 ℃～32 ℃ vibration inducing culture 10～25h, shaking speed 150～220r/min; Finally add glycine, under the same terms, continue vibration inducing culture 20～40h, ferment complete, obtain fermented liquid;
(3) extraction of crude enzyme liquid: step (2) gained fermented liquid is centrifugal; Collect supernatant liquor, be the outer crude enzyme liquid of born of the same parents; Collect somatic cells resuspended rear ultrasonication of Tris-HCl for precipitation, gained ultrasonication liquid is centrifugal, collect supernatant, be crude enzyme liquid in born of the same parents.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: the described seed culture based component of step (1) is counted by grams per liter: peptone 8～12g, yeast powder 3～10g, NaCl8～12g, all the other compositions are water, pH7.2～7.6.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: the described fermentation culture based component of step (2) is counted by grams per liter: peptone 8～12g, yeast powder 5～25g, glycerine 0～6g, all the other compositions are 0.25mol/L Tris-HCl(pH7.2).
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: the addition of the described lactose of step (2) is every liter of described fermention medium 1～10g.
As a kind of preferred version of preparing the method for Phospholipase C of the present invention, wherein: the addition of the described glycine of step (2) is every liter of described fermention medium 0～5g.
Compare with wild isolated strains and the engineering strain of existing product Phospholipase C, the present invention has built a strain goal gene and has derived from listerisa monocytogenes in mjme (L.monocytogenes), genetically engineered recombinant escherichia coli BL21(DE3 that can high yield Phospholipase C)/pET-lm-plc CCTCC No.M2013301, utilize this bacterial strain to produce Phospholipase C by liquid fermenting, yield of enzyme is high, its enzymic activity reaches as high as 779.358U/ml, and security is good; This enzyme method production technique processed is simple, with short production cycle, and cost is low, at raw plant oil, has certain application prospect aspect coming unstuck.
The measuring method that recombination phospholipase C enzyme is lived is as follows:
P-NPPC standard measure is measured enzyme and is lived.P-NPPC is a kind of substrate structure analogue of Yelkin TTS, PLC hydrolyzable p-NPPC generates p-NP, p-NP is a kind of yellow substance, at 410nm place, there is maximum absorption band, utilize spectrophotometry fermented liquid at the light absorption value at 410nm place, can reflect that PLC hydrolysis p-NPPC produces the amount of p-NP, according to p-NP typical curve, can quantitative Analysis go out corresponding enzyme activity size; Enzyme reaction composing system is as follows: 50mM/L Tris-HCl (pH7.2), 10mmol/L NPPC.In 1mL reaction system, add the fermented liquid of 100 μ L, in 37 ℃ of reaction 30min; Enzyme activity unit is defined as follows: at pH7.2, temperature is under the condition of 37 ℃, and the amount that per minute hydrolysis p-NPPC produces the required enzyme of 1nmol p-NP is 1 enzyme activity unit (U).
A further object of the present invention is to provide raw plant oil, as soybean oil, rapeseed oil, rice bran wet goods carry out the method for degumming process, the method not only can effectively reduce the phosphorus content in crude oil, also can improve the content of triglyceride in processed oil, improve the yield of neutral oil, saved cost.
According to a further aspect of the invention, the invention provides a kind of method that raw plant oil comes unstuck, comprise, the preheating of raw plant oil and sour pre-treatment; Add Phospholipase C as claimed in claim 3 to react; The enzyme that goes out, centrifugation completes comes unstuck.
A kind of preferred version of the method for coming unstuck as raw plant oil of the present invention, wherein: concrete steps are as follows:
(1), by raw plant oil heating in water bath to 60～80 ℃ in tool plug Erlenmeyer flask, adding quality is that the mass concentration of total quality of crude oil 0.5% is 25%～50% citric acid solution, carries out the sour pre-treatment of 15～25min under 300～500r/min agitation condition.
(2) will be cooled to 40～60 ℃ through the pretreated oil sample of peracid, and add a certain amount of 4%NaOH solution to mix to regulate pH to 4～7.
The mensuration of oil phase pH: get oil-water mixture 40g with 50mL centrifuge tube, centrifugal 10min under 5000r/min condition, discard upper oil phase, in precipitation, add the distilled water of 5mL again, centrifugal 10min under 5000r/min condition again after being fully uniformly mixed, get water in centrifuge tube and measure pH value, calibrated rear employing with pH meter.Experience updating formula when oil phase pH is 5.0 left and right: pH reality=pH measures-0.3.
(3) add again oil to weigh 1%～3% distilled water, and the described Phospholipase C of 400～2000U/Kg, evenly mixing, 300r/min at 40 ℃～75 ℃～500r/min stirs 1～2h, carries out enzyme reaction.
(4) reaction system is warming up to 90 ℃ of enzyme 10min that go out above, at 8000～10000r/min, carries out 10min centrifugation, complete described Phospholipase C the catalysis of raw plant oil is come unstuck.
Utilize this recombination phospholipase C to raw plant oil, as soybean oil, rapeseed oil, rice bran wet goods carry out degumming process, not only can effectively reduce the phosphorus content in crude oil, also can improve the content of triglyceride in processed oil, improve crude oil yield, cost-saving, to Oils and fats enterprise, brought huge interests.
Embodiment
A lot of details have been set forth 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 and implement, those skilled in the art can do similar popularization without prejudice to intension of the present invention in the situation that, so the present invention is not subject to the restriction of following public specific embodiment.
In following embodiment the experiment material of using as follows: expression vector pET-28a (+) and e. coli jm109, BL21 (DE3) are preserved by this laboratory; Listerisa monocytogenes in mjme (L.monocytogenes) Chinese industrial microbial strains preservation administrative center (CICC), deposit number is: 21540, pMD18T-simple Vector, T4DNA ligase enzyme and 10 * T4 ligase enzyme Buffer are purchased from precious biotechnology (Dalian) company limited; Restriction enzyme Not I and EcoR I are purchased from BioLabs.
The clone of embodiment 1 phospholipase C gene
According to the primers of the plc gene (YP005964174.1) of the L.monocytogenes providing on NCBI, wherein, upstream primer is:
5 '-CG
gAATTCaTGTGTTGTGATGAATACTTACAAA-3 ' (underscore marking part divides and represents EcoR I restriction enzyme site);
Downstream primer is:
5 '-AT
gCGGCCGCtTATTCATTTGTTTTTTT-3 ' (underscore marking part divides and represents NotI restriction enzyme site).
The genomic dna of the listerisa monocytogenes in mjme (L.monocytogenes) that the deposit number of take is CICC No.21540 is template, use the method clone of PCR to obtain hemolytic phospholipase C gene, and be connected with carrier pMD18T-simple and build cloning vector pMD18T-simple-lm-plc, this cloning vector is transformed to competent escherichia coli cell E.coli JM109, select positive colony, sequence verification, its base sequence is as shown in SEQ ID NO:1, and result shows that phospholipase C gene clones successfully.
The structure of embodiment 2 recombinant plasmid pET-L.m-plc
With EcoR I and Not I to recombinant vectors pMD18T-simple-lm-plc and expression vector pET-28a(+) carry out double digestion, endonuclease reaction system is 20 μ L: plasmid 10 μ L, Buffer32 μ L, Not I0.5 μ L, EcoR I0.5 μ L, 100*Bsa0.2 μ L, H206.8 μ L.Reclaim goal gene and carrier DNA, and connect with T4DNA ligase enzyme, ligation system 10 μ L: goal gene 6 μ L, carrier DNA 1.2 μ L, 10 * T4 ligase enzyme Buffer1 μ L, T4DNA ligase enzyme 1 μ L, H200.8 μ L, in 16 ℃ of reaction 12h.
To connect product and transform competent escherichia coli cell E.coli JM109, method for transformation is as follows:
(1) in the E.coli JM109 competent cell (100ul/ pipe) of packing, add the above-mentioned connection product of 10ul and mix gently, ice bath 30min;
(2) above-mentioned EP pipe is placed in to 42 ℃ of water-baths, accurately timing 90s, does not shake EP pipe;
(3) shift EP pipe in ice bath, cooling 3min;
(4) every pipe adds aseptic TB substratum 890 μ L, is placed in 37 ℃ of shaking tables, 100r/min, recovery 1h;
(5) with the centrifugal 2min of 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 10～18h in 37 ℃ of incubators;
(6) picking positive colony, and through Not I and EcoR I double digestion checking recombinant plasmid pET-L.m-plc.
(7) get successfully the glycerine that the recombinant plasmid pET-L.m-plc that builds and isopyknic volume fraction are 30% and mix, in-80 ℃ of preservations.
Embodiment 3 recombination bacillus coli BL21(DE3)-pET28a-L.m-PLC, deposit number is the structure of CCTCC No.M2013301
The recombinant plasmid pET-L.m-plc building is transformed to e. coli bl21 (DE3), and method for transformation is as follows:
(1) to the E.coli BL21(DE3 of packing) add the above-mentioned recombinant plasmid pET-lm-plc of 0.5ul and mix gently, ice bath 30min in 100 μ L competent cells (100ul/ pipe);
(2) above-mentioned EP pipe is placed in to 42 ℃ of water-baths, accurately timing 90s, does not shake EP pipe;
(3) shift EP pipe in ice bath, cooling 3min;
(4) every pipe adds aseptic TB substratum 890 μ L, is placed in 37 ℃ of shaking tables, 100r/min, recovery 1h;
(5) with the centrifugal 2min of 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 10～18h in 37 ℃ of incubators;
(6) picking positive colony, Not I and EcoR I double digestion checking recombination bacillus coli BL21(DE3)-pET28a-L.m-PLC.
(7) getting successfully the recombination bacillus coli BL21(DE3 building)-pET28a-L.m-PLC and isopyknic volume fraction be 30% glycerine mixes, in-80 ℃ of preservations.
Embodiment 4 recombination bacillus coli BL21(DE3)-pET28a-L.m-PLC prepares Phospholipase C
Use recombination bacillus coli BL21(DE3)-pET28a-L.m-PLC deposit number is that CCTCC No.M2013301 prepares Phospholipase C.
(1) seed culture: the recombination bacillus coli BL21(DE3 that gets the above-mentioned preservation of 100 μ L)-pET28a-L.m-PLC, CCTCC No.M2013301 is inoculated in seed culture medium, on Clothoid type shaking table in 37 ℃ of shaking culture 10h, shaking speed 200r/min; Above-mentioned seed culture based component is counted by grams per liter: peptone 10g, and yeast powder 5g, NaCl10g, all the other compositions are water, in 1 * 10
5sterilizing 20min under Pa high pressure.
(2) liquid fermentation and culture: by through step (1), cultivate gained activated seed liquid by volume the inoculum size of per-cent 5% be seeded to fermention medium, fermention medium is loaded on 250ml triangular flask, liquid amount is 50ml, on Clothoid type shaking table in 35 ℃ of shaking culture 4h hour, shaking speed 200r/min; Adding lactose to final concentration is 2.5g/L, and on Clothoid type shaking table, in 30 ℃ of vibration inducing culture 20h, shaking speed is 200r/min; Ferment complete, obtain fermented liquid; Above-mentioned fermentation culture based component is counted by grams per liter: peptone 10g, and yeast powder 5g, glycerine 2g, all the other compositions are 0.25mol/L Tris-HCl(pH7.2), sterilizing 20min under 1 * 105Pa high pressure.
(3) extraction of crude enzyme liquid: by step (2) gained fermented liquid in 4 ℃ of centrifugal 10min, rotating speed 10000r/min; Collection somatic cells precipitation, uses 25mmol/L Tris-HCl(pH7.2) resuspended thalline, be placed in Ultrasonic Cell Disruptor ultrasonication 10min, gained ultrasonication liquid is centrifugal, get supernatant, be crude enzyme liquid in born of the same parents.The enzyme work that utilizes p-NPPC standard measure to record crude enzyme liquid in born of the same parents reaches 376.026U/ml.
Embodiment 5 recombination bacillus coli BL21(DE3)-pET28a-L.m-PLC prepares Phospholipase C
(1) seed culture: the recombination bacillus coli BL21(DE3 that gets the above-mentioned preservation of 100 μ L)-pET28a-L.m-PLC, CCTCC No.M2013301 is inoculated in seed culture medium, on Clothoid type shaking table in 32 ℃ of shaking culture 14h, shaking speed 200r/min; Above-mentioned seed culture based component is counted by grams per liter: peptone 10g, and yeast powder 6g, NaCl10g, all the other compositions are water, in 1 * 10
5sterilizing 20min under Pa high pressure.
(2) liquid fermentation and culture: by through step (1), cultivate gained activated seed liquid by volume the inoculum size of per-cent 5% be seeded to fermention medium, fermention medium is loaded on 250ml triangular flask, liquid amount is 50ml, on Clothoid type shaking table in 30 ℃ of shaking culture 8h hour, shaking speed 150r/min; Adding lactose to final concentration is 5g/L, and on Clothoid type shaking table, in 28 ℃ of vibration inducing culture 14h, shaking speed is 220r/min; Adding glycine to final concentration is 1g/L again, continues vibration inducing culture 10h under the same terms; Ferment complete, obtain fermented liquid; Above-mentioned fermentation culture based component is counted by grams per liter: peptone 9g, and yeast powder 12g, glycerine 3g, all the other compositions are 0.25mol/L Tris-HCl(pH7.2), in 1 * 10
5sterilizing 20min under Pa high pressure.
(3) extraction of crude enzyme liquid: in 4 ℃ of centrifugal 10min, rotating speed 10000r/min, collects supernatant liquor by step (2) gained fermented liquid, is the outer crude enzyme liquid of born of the same parents; Collection somatic cells precipitation, with 25mmol/LTris-HCl(pH7.2) resuspended thalline, be placed in Ultrasonic Cell Disruptor ultrasonication 10min, gained ultrasonication liquid is centrifugal, get supernatant, be crude enzyme liquid in born of the same parents; Utilize p-NPPC standard measure to record total enzyme work of crude enzyme liquid in born of the same parents and outside born of the same parents and reach 503.821U/ml.
Embodiment 6
(1) seed culture: the recombination bacillus coli BL21(DE3 that gets the above-mentioned preservation of 50 μ L)-pET28a-L.m-PLC, CCTCC M2013301 is inoculated in seed culture medium, on Clothoid type shaking table in 35 ℃ of shaking culture 12h, shaking speed 180r/min; Above-mentioned seed culture based component is counted by grams per liter: peptone 11g, and yeast powder 6g, NaCl11g, all the other compositions are water, in 1 * 10
5sterilizing 20min under Pa high pressure.
(2) liquid fermentation and culture: by through step (1), cultivate gained activated seed liquid by volume the inoculum size of per-cent 5% be seeded to fermention medium, fermention medium is loaded on 250ml triangular flask, liquid amount is 50ml, on Clothoid type shaking table in 37 ℃ of shaking culture 6h hour, shaking speed 180r/min; Adding lactose to final concentration is 1g/L, and on Clothoid type shaking table, in 25 ℃ of vibration inducing culture 20h, shaking speed is 200r/min; Adding glycine to final concentration is 3g/L again, continues vibration inducing culture 18h under the same terms; Ferment complete, obtain fermented liquid; Above-mentioned fermentation culture based component is counted by grams per liter: peptone 12g, and yeast powder 22g, glycerine 4g, all the other compositions are 0.25mol/L Tris-HCl(pH7.2), in 1 * 10
5sterilizing 20min under Pa high pressure.
(3) extraction of crude enzyme liquid: with embodiment 5
Utilize p-NPPC standard measure to record total enzyme work of crude enzyme liquid in born of the same parents and outside born of the same parents and reach 779.358U/ml.
The crude rapeseed oil of embodiment 7 restructuring PLC comes unstuck
Crude rapeseed oil is heated to 80 ℃, and adding mass concentration 45%, quality is the citric acid solution of crude rapeseed oil 0.5%, carries out the sour pre-treatment of 20min under 500r/min agitation condition.To be cooled to 47 ℃ through the pretreated oil sample of peracid, add a certain amount of 4%NaOH solution, mix to regulate pH to 5.2.Add oil to weigh 1% distilled water and 500U/kg recombination phospholipase C, evenly mix, at 62 ℃, 300r/min stirring 1h carries out enzyme reaction.After reaction finishes, reaction system is warming up to 90 ℃ of enzyme 10min that go out, at 8000r/min, carries out 10min centrifugation, complete recombination phospholipase C the catalysis of crude rapeseed oil is come unstuck.
Through present embodiment, crude rapeseed oil is carried out after degumming process, the phosphorus content of the rapeseed oil that comes unstuck is 4.503mg/kg, before phosphorus content ratio comes unstuck, has reduced by 97.55% left and right.
The rice bran degumming raw oil of embodiment 8 restructuring PLC
Rice bran crude oil is heated to 80 ℃, and adding mass concentration 50%, quality is the citric acid solution of rice bran crude oil 0.45%, carries out the sour pre-treatment of 20min under 500r/min agitation condition.To be cooled to 47 ℃ through the pretreated oil sample of peracid, add a certain amount of 4%NaOH solution, mix to regulate pH to 5.0.Add oil to weigh 1% distilled water and the recombination phospholipase C of 1000U/kg, evenly mix, at 60 ℃, 300r/min stirs 1h and carries out enzyme reaction.After reaction finishes, reaction system is warming up to 90 ℃ of enzyme 10min that go out, at 10000r/min, carries out 10min centrifugation, complete recombination phospholipase C the catalysis of rice bran crude oil is come unstuck.
Through present embodiment, rice bran crude oil is carried out after degumming process, the phosphorus content of the Rice pollard oil that comes unstuck is 50.191mg/kg, before phosphorus content ratio comes unstuck, has reduced by 83.63% left and right.
The crude oil of soybean of embodiment 9 restructuring PLC comes unstuck
Crude oil of soybean is heated to 80 ℃, and adding mass concentration 45%, quality is the citric acid solution of crude oil of soybean 0.5%, carries out the sour pre-treatment of 20min under 500r/min agitation condition.To be cooled to 47 ℃ through the pretreated oil sample of peracid, add a certain amount of 4%NaOH solution, mix to regulate pH to 5.2.Add oil to weigh 1% distilled water and 500U/kg recombination phospholipase C, evenly mix, at 62 ℃, 300r/min stirring 1.2h carries out enzyme reaction.After reaction finishes, reaction system is warming up to 90 ℃ of enzyme 10min that go out, at 10000r/min, carries out 10min centrifugation, complete recombination phospholipase C the catalysis of crude oil of soybean is come unstuck.
Through present embodiment, crude oil of soybean is carried out after degumming process, the phosphorus content of the soybean oil of coming unstuck is 3.945mg/kg, before phosphorus content ratio comes unstuck, has reduced by 98.29% left and right.
The crude oil of soybean of embodiment 10 restructuring PLC comes unstuck
Crude oil of soybean is heated to 80 ℃, and adding mass concentration 45%, quality is the citric acid solution of crude oil of soybean 0.5%, carries out the sour pre-treatment of 20min under 500r/min agitation condition.To be cooled to 47 ℃ through the pretreated oil sample of peracid, add a certain amount of 4%NaOH solution, mix to regulate pH to 4.5.Add oil to weigh 1% distilled water and 600U/kg recombination phospholipase C, evenly mix, at 67 ℃, 300r/min stirring 1h carries out enzyme reaction.After reaction finishes, reaction system is warming up to 90 ℃ of enzyme 10min that go out, at 10000r/min, carries out 10min centrifugation, complete recombination phospholipase C the catalysis of crude oil of soybean is come unstuck.
Through present embodiment, crude oil of soybean is carried out after degumming process, the phosphorus content of the soybean oil of coming unstuck is 3.705mg/kg, before phosphorus content ratio comes unstuck, has reduced by 98.40% left and right, and in soybean oil, diglyceride content ratio increases approximately 0.8% before coming unstuck.
It should be noted that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (10)
1. recombination bacillus coli BL21 (DE3)-pET28a-L.m-PLC, deposit number is CCTCC No.M2013301.
2. recombination bacillus coli BL21 (DE3)-pET28a-L.m-PLC according to claim 1, CCTCC No.M2013301, is characterized in that: by following method, made:
(1) take the genome of the listerisa monocytogenes in mjme (L.monocytogenes) that deposit number is CICC No.21540 is template, and clone obtains phospholipase C gene, and its base sequence is as shown in SEQ ID NO:1;
(2) step (1) gained phospholipase C gene is cloned into expression vector pET-28a(+) upper, obtain recombinant vectors;
(3) step (2) gained recombinant vectors is transformed to competent escherichia coli cell, build and obtain recombination bacillus coli.
3. with recombination bacillus coli BL21(DE3 described in claim 1 or 2)/pET-lm-plc, CCTCC No.M2013301 prepares the method for Phospholipase C, it is characterized in that: take this recombination bacillus coli carries out liquid fermenting as fermentation strain, prepares Phospholipase C.
4. prepare according to claim 3 the method for Phospholipase C, it is characterized in that: comprise,
(1) seed culture: by described recombination bacillus coli BL21 (DE3)-pET28a-L.m-PLC, CCTCC No.M2013301 is inoculated in seed culture medium, in 28 ℃～37 ℃ shaking culture 6h～14h, shaking speed 150～220r/min;
(2) liquid fermentation and culture: by through step (1), cultivate gained activated seed liquid by volume the inoculum size of per-cent 1～10% be seeded to fermention medium, in 28～37 ℃ of shaking culture 4h～6h hour, shaking speed 150～220r/min; Add again lactose, in 18 ℃～32 ℃ vibration inducing culture 10～25h, shaking speed 150～220r/min; Finally add glycine, under the same terms, continue vibration inducing culture 20～40h, ferment complete, obtain fermented liquid;
(3) extraction of crude enzyme liquid: step (2) gained fermented liquid is centrifugal; Collect supernatant liquor, be the outer crude enzyme liquid of born of the same parents; Collect somatic cells resuspended rear ultrasonication of Tris-HCl for precipitation, gained ultrasonication liquid is centrifugal, collect supernatant, be crude enzyme liquid in born of the same parents.
5. prepare according to claim 4 the method for Phospholipase C, it is characterized in that: the described seed culture based component of step (1) is counted by grams per liter: peptone 8～12g, yeast powder 3～10g, NaCl8～12g, all the other compositions are water, pH7.2～7.6.
6. prepare according to claim 4 the method for Phospholipase C, it is characterized in that: the described fermentation culture based component of step (2) is counted by grams per liter: peptone 8～12g, yeast powder 5～25g, glycerine 0～6g, all the other compositions are 0.25mol/L Tris-HCl(pH7.2).
7. prepare according to claim 4 the method for Phospholipase C, it is characterized in that: the addition of the described lactose of step (2) is every liter of described fermention medium 1～10g.
8. prepare according to claim 4 the method for Phospholipase C, it is characterized in that: the addition of the described glycine of step (2) is every liter of described fermention medium 0～5g.
9. the method that raw plant oil comes unstuck, is characterized in that: comprises,
The preheating of raw plant oil and sour pre-treatment;
Add Phospholipase C as claimed in claim 3 to react;
The enzyme that goes out, centrifugation completes comes unstuck.
10. the method that raw plant oil comes unstuck according to claim 9, is characterized in that: concrete steps are as follows:
(1), by raw plant oil heating in water bath to 60～80 ℃ in tool plug Erlenmeyer flask, adding quality is that the mass concentration of total quality of crude oil 0.5% is 25%～50% citric acid solution, carries out the sour pre-treatment of 15～25min under 300～500r/min agitation condition.
(2) will be cooled to 40～60 ℃ through the pretreated oil sample of peracid, and add a certain amount of 4%NaOH solution to mix to regulate pH to 4～7.
(3) add again oil to weigh 1%～3% distilled water, and the described Phospholipase C of 400～2000U/Kg, evenly mixing, 300r/min at 40 ℃～75 ℃～500r/min stirs 1～2h, carries out enzyme reaction.
(4) reaction system is warming up to 90 ℃ of enzyme 10min that go out above, at 8000～10000r/min, carries out 10min centrifugation, complete described Phospholipase C the catalysis of raw plant oil is come unstuck.
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| CN105734029A (en) * | 2014-12-12 | 2016-07-06 | 丰益(上海)生物技术研发中心有限公司 | Phospholipase antibacterial peptide |
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| CN106119237A (en) * | 2016-08-05 | 2016-11-16 | 集美大学 | A kind of method of modifying of recombination phospholipase C |
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| CN107245470B (en) * | 2017-05-24 | 2020-12-22 | 华南理工大学 | Lipase recombinant escherichia coli expression strain, recombinant lipase and application |
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