CN103525744A - Recombinant escherichia coli, method for preparing phospholipase C and application - Google Patents

Abstract

The invention discloses a recombinant Escherichia coli and a method and application for preparing phospholipase C. The target gene used is derived from the recombinant Escherichia coli BL21(DE3)-pET28a- L.m-PLC is used as a fermentation strain to carry out liquid fermentation to prepare phospholipase C. The enzyme production process is simple, the production cycle is short, and the cost is low. And the recombinant phospholipase C can specifically hydrolyze the glycerophosphate bond on the C3 position of glycerophospholipid to produce DAG, and use it to degumming vegetable crude oil (soybean oil, rapeseed oil, rice bran oil, etc.).

Description

A kind of recombinant escherichia coli and the method and application of preparing phospholipase C

technical field

The invention relates to a production method of phospholipase C derived from Listeria monocytogenes and its application in the oil degumming industry, belonging to the fields of enzyme gene engineering and enzyme engineering. the

Background technique

Listeria monocytogenes (L.monocytogenes) is an important food-borne pathogen, the growth temperature ranges from -1.5°C to 45°C, and the optimum temperature is 30°C to 37°C. Listeria monocytogenes is a typical cold-resistant bacterium that can grow and multiply in the cold room of ordinary refrigerators, and can still partially survive at -20°C, and the longer the food containing Listeria monocytogenes is refrigerated, the greater the danger ; and has strong heat resistance, milk pasteurization temperature can not kill it. Facultative anaerobic, often grows in a microaerophilic environment containing CO2; does not require high nutrition, can form biofilm on the surface of objects, is resistant to antibacterial substances, and is also acid-resistant, resistant to repeated freezing and thawing, and anti-drying. It is salt tolerant. Listeria monocytogenes can grow in wild animals, livestock, birds, insects, sewage, soil and plants, therefore, Listeria monocytogenes is often detected in cattle, sheep, poultry, dairy products, fruits Pollution of varying degrees often occurs in vegetables, vegetables, cooked meat products, and seafood. Clinically, listeriosis infection includes miscarriage, sepsis, meningitis, etc. The symptoms are similar to influenza, and are often accompanied by febrile gastroenteritis syndrome. the

Listeria monocytogenes can produce two phospholipase C (PLC): phosphatidylinositol phospholipase C (PI-PLC) and phosphatidylcholine phospholipase C (PC-PLC). The former is encoded by the plcA gene, and the latter is encoded by the plcB gene. Both plcA gene and plcB gene are important gene components of virulence island 1 (LIPI-1), which is related to the intracellular parasitic life cycle of Listeria monocytogenes. the

PLC mainly acts on the glycerophosphate bond at the C3 position of glycerophospholipids, and the hydrolysis products are diacylglycerol (DAG) and phosphatidic acid compounds (phosphocholine, phosphoethanolamine, phosphoserine, and phosphoinositide, etc.), which have always played an important role in the pharmaceutical industry. application. At present, because the hydrolyzed product DAG can become a component of oil together with triglyceride (TAG) and does not need to be removed, it has attracted the attention of the oil industry, and is being used more and more as a new type of phospholipase for enzymatic degumming. Used in grease degumming process. PLC enzymatic degumming has the common advantages of general enzymatic degumming, such as wide applicability, mild reaction conditions, saving the consumption of acid and alkali chemicals in production, almost no soapstock and waste water, etc., and obvious growth in economic benefits, but compared with other Advanced enzymatic degumming, such as phospholipase A (PLA) enzymatic degumming, PLC enzymatic degumming can not only greatly shorten the degumming time, but also effectively increase the yield of crude oil and reduce the loss of crude oil. Usually, every 500ppm phosphorus can increase about 1 % oil production, this feature is particularly important today when food prices continue to rise. the

（PLC），已经成为目前工艺最成熟的脱胶用PLC产品。PLC（BD16449）是Vince Ciofalo利用毕赤酵母SMD1168重组表达的一种分子量约为34kDa的糖基化蛋白酶，安全性高，被应用于植物油脱胶。2008年，C.L.g.戴顿等在Gramatikova的研究基础上，综合考虑PLA和PLC的优缺点，发明了PC-PLC/PI-PLC与PLA1/PLA2的混合制剂，用以去除植物油中的磷脂。研究表明，复合制剂的酶促反应速度明显优于单独使用，可以讲残磷含量降至3ppm以下，从而避免了其他辅助脱胶工序，节省了大量的化学试剂和水。Danisco也通过

（LAT）和PLC的复合制剂对胶质进行处理，显著减少了毛油的炼耗率。  The research on phospholipase C for degumming started earlier abroad. In 1988, Graille et al. proposed to use PLC to treat oil and convert phospholipids into DAG, which not only does not take away the TAG component, but also produces some "oil" to reduce the loss of oil during the refining process. Graille et al. further studied the use of Bacillus cereus to prepare PLC enzyme preparations, but due to the limitation of preparation costs at that time, the potential commercial value of PLC was ignored. In 2005, S. Gramatikova expressed heterologous PLC with Pichia pastoris strain of Mut+ phenotype for oil treatment, which can reduce the phospholipid residue in crude oil to below 10ppm. However, since most PLCs belong to phosphatidylcholine-specific phospholipase C (PC-PLC), they have high specificity for phospholipase choline (PC) and phosphatidylethanolamine (PE), while for phosphatidylinositol (PI) has low activity, and only PC-PLC degumming cannot completely remove phosphorus in crude oil. Therefore, Gramatikova first proposed the combined use of PLC and PLA for oil degumming in 2005. In 2006, Nagasaki et al. purified a new type of PLC by using Aspergillus luteus IAM13907 strain and Aspergillus oryzae NBRC4190 strain. The relative molecular weight is about 87kDa, and it is relatively stable in the range of 0-80°C. It is widely used in the food and cosmetic industries. In 2007, Verenium launched at the AOCS annual meeting

(PLC), has become the most mature PLC product for degumming at present. PLC (BD16449) is a glycosylated protease with a molecular weight of about 34kDa recombinantly expressed by Vince Ciofalo using Pichia pastoris SMD1168. It has high safety and is used in vegetable oil degumming. In 2008, based on Gramatikova's research, CLg Dayton et al. considered the advantages and disadvantages of PLA and PLC, and invented a mixed preparation of PC-PLC/PI-PLC and PLA1/PLA2 to remove phospholipids in vegetable oil. Studies have shown that the enzymatic reaction speed of the compound preparation is significantly better than that of the single use, and the residual phosphorus content can be reduced to below 3ppm, thereby avoiding other auxiliary degumming processes and saving a lot of chemical reagents and water. Danisco also passed

The compound preparation of (LAT) and PLC treats the colloid, which significantly reduces the refining consumption rate of crude oil.

Domestic research on PLC for oil degumming is still in its infancy. Chen Tao’s team from Wuhan Institute of Virology has been committed to the research of microbial PLC. Li Caifeng and Zhan Yishu screened PLC-producing Vibrio harveii and Bacillus cereus respectively. Liu Feifei used Escherichia coli and Pichia pastoris to recombinantly express Bacillus cereus PLC , Zhao Jinxing recombinantly expressed Pseudomonas aeruginosa PLC using Escherichia coli, but none of the prepared PLC was applied to oil degumming. In 2003, Sun Chunlai isolated and purified a PLC from the fermentation supernatant of Serratia marcescens. In 2006, Meng Qingfei used this enzyme to degumming soybean oil, so that the hydrolysis rate of phospholipids in crude oil reached 85.7%, and the refining rate increased by 3.08%. Yang Jiao used PLC to degumming crude soybean oil, reducing the residual phosphorus to 17.8ppm. In 2012, Yu Dianyu et al. used sodium alginate and chitosan to immobilize PLC to degumming soybean crude oil, reducing the phosphorus content to 3.8ppm. In 2013, Liu Lu and others used PLC to degumming rapeseed crude oil, reducing the phosphorus content to 4.3ppm. the

Although the application of PLC enzymatic degumming technology in the oil industry started relatively late, it has developed rapidly in recent years. Therefore, it is of great practical significance to develop a low-cost, high-quality, and versatile PLC for enzymatic degumming with my country's independent property rights. the

Contents of the invention

In view of the above and/or existing problems in the field of enzyme genetic engineering and enzyme engineering, the present invention is proposed. the

Therefore, one object of the present invention is to provide a recombinant Escherichia coli. the

In order to solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solution: a recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC, the preservation number is CCTCC No.M2013301. the

As a construction scheme of recombinant escherichia coli BL21(DE3)-pET28a-L.m-PLC of the present invention, wherein: prepared by the following method:

(1) Using the genome of Listeria monocytogenes (L.monocytogenes) with the preservation number CICC No. 21540 as a template, the phospholipase C gene was cloned, and its base sequence is shown in SEQ ID NO: 1;

(2) Cloning the phospholipase C gene obtained in step (1) into the expression vector pET-28a(+) to obtain a recombinant vector;

(3) Transform Escherichia coli competent cells with the recombinant vector obtained in step (2) to construct recombinant Escherichia coli. the

The Escherichia coli (Escherichia coli) BL21(DE3)-pET28a-L.m-PLC provided by the present invention has been preserved in the China Center for Type Culture Collection (CCTCC) on June 28, 2013, and the preservation number is: CCTCC No.M2013301, Address: Wuhan University, Wuhan, China. The strain is hereinafter referred to as: Escherichia coli (Escherichia coli) BL21 (DE3)-pET28a-L.m-PLC. the

Another object of the present invention is to provide a method for preparing phospholipase C by using recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC as a fermentation strain for liquid fermentation. The enzyme production process is simple, the production cycle is short, and the cost is low , has a certain application prospect in the degumming of vegetable crude oil. the

As a preferred version of the method for preparing phospholipase C according to the present invention, wherein: comprising,

(1) Seed culture: inoculate the recombinant Escherichia coli BL21(DE3)/pET-lm-plc, CCTCC No.M2013301 in the seed medium, and culture it with shaking at 28°C-37°C for 6h-14h, the shaker speed is 150r /min～220r/min;

(2) Liquid fermentation culture: inoculate the fermentation medium with the inoculum amount of 1-10% by volume of the activated seed liquid obtained through the cultivation in step (1), shake and culture at 28-37°C for 4h-6h, and shaker speed 150 ~220r/min; then add lactose, shake induction culture at 18℃~32℃ for 10~25h, shaker speed 150~220r/min; finally add glycine, continue shaking induction culture under the same conditions for 20~40h, the fermentation is complete, get fermented liquid;

(3) Extraction of crude enzyme solution: Centrifuge the fermented solution obtained in step (2); collect the supernatant, which is the extracellular crude enzyme solution; collect the bacterial cell pellet and resuspend it with Tris-HCl, then ultrasonically break it, and ultrasonicate the obtained Centrifuge the crushed solution, collect the supernatant, which is the intracellular crude enzyme solution. the

As a preferred scheme of the method for preparing phospholipase C according to the present invention, wherein: the composition of the seed medium in step (1) is calculated in grams per liter: peptone 8-12g, yeast powder 3-10g, NaCl 8-12g , the remaining ingredients are water, pH7.2～7.6. the

As a preferred version of the method for preparing phospholipase C in the present invention, wherein: the components of the fermentation medium in step (2) are calculated in grams per liter: peptone 8-12g, yeast powder 5-25g, glycerin 0-25g 6g, and the remaining components are 0.25mol/L Tris-HCl (pH7.2). the

As a preferred solution of the method for preparing phospholipase C in the present invention, wherein: the amount of lactose added in step (2) is 1-10 g per liter of the fermentation medium. the

As a preferred solution of the method for preparing phospholipase C in the present invention, wherein: the added amount of glycine in step (2) is 0-5 g per liter of the fermentation medium. the

Compared with the existing wild isolates and genetically engineered strains producing phospholipase C, the present invention constructs a genetic engineering strain whose target gene is derived from L. monocytogenes (L. Recombinant Escherichia coli BL21 (DE3)/pET-lm-plc CCTCC No.M2013301, using this strain to produce phospholipase C through liquid fermentation, has high enzyme production, and its enzyme activity can reach up to 779.358U/ml, and it is safe The enzyme preparation method has simple production process, short production cycle and low cost, and has certain application prospects in the degumming of vegetable crude oil. the

The assay method of recombinant phospholipase C enzyme activity is as follows:

Enzyme activity was quantified by p-NPPC method. p-NPPC is a substrate structure analogue of lecithin, PLC can hydrolyze p-NPPC to generate p-nitrophenol, which is a yellow substance with a maximum absorption peak at 410nm, which can be determined by spectrophotometry The absorbance value of the fermentation broth at 410nm can reflect the amount of p-nitrophenol produced by PLC hydrolysis of p-NPPC, and the corresponding enzyme activity can be quantitatively calculated according to the p-nitrophenol standard curve; the composition of the enzyme reaction system is as follows: 50mM/L Tris -HCl (pH7.2), 10mmol/L NPPC. Add 100 μL of fermentation broth to 1 mL of reaction system, and react at 37°C for 30 minutes; the enzyme activity unit is defined as follows: at pH 7.2 and temperature at 37°C, the hydrolysis of p-NPPC per minute required to produce 1 nmol of p-nitrophenol The amount of enzyme is 1 enzyme activity unit (U). the

Another object of the present invention is to provide a method for degumming vegetable crude oil, such as soybean oil, rapeseed oil, rice bran oil, etc., which can not only effectively reduce the phosphorus content in crude oil, but also increase the glycerol content in the finished oil. The content of diester improves the yield of neutral oil and saves cost. the

According to another aspect of the present invention, the present invention provides a method for degumming vegetable crude oil, comprising, preheating and acid pretreatment of vegetable crude oil; adding phospholipase C as claimed in claim 3 to react; inactivating enzyme, Centrifugation completes degumming. the

As a preferred version of the method for vegetable crude oil degumming of the present invention, wherein: the specific steps are as follows:

(1) Heat vegetable crude oil in a water bath in a stoppered Erlenmeyer flask to 60-80°C, add a citric acid solution with a mass concentration of 0.5% of the total crude oil mass and a mass concentration of 25%-50%, and stir at 300-500r/min 15-25 minutes of acid pretreatment under the conditions. the

(2) Cool the acid-pretreated oil sample to 40-60°C, add a certain amount of 4% NaOH solution and mix well to adjust the pH to 4-7. the

Determination of the pH of the oil phase: take 40g of the oil-water mixture in a 50mL centrifuge tube, centrifuge at 5000r/min for 10min, discard the upper oil phase, then add 5mL of distilled water to the precipitate, stir and mix thoroughly, and again under the condition of 5000r/min Centrifuge for 10 minutes, take the water phase in the centrifuge tube and measure the pH value with a pH meter, and use it after correction. The empirical correction formula when the pH of the oil phase is around 5.0: actual pH = measured pH - 0.3. the

(3) Add distilled water with an oil weight of 1% to 3%, and the phospholipase C at 400 to 2000 U/Kg, mix evenly, and stir at 300r/min to 500r/min at 40°C to 75°C for 1 to 2 hours, Perform an enzymatic reaction. the

(4) Elevate the temperature of the reaction system to above 90° C. to inactivate the enzyme for 10 minutes, and perform centrifugation at 8,000 to 10,000 r/min for 10 minutes to complete the catalyzed degumming of vegetable crude oil by the phospholipase C. the

Using the recombinant phospholipase C to degumming crude vegetable oil, such as soybean oil, rapeseed oil, rice bran oil, etc., can not only effectively reduce the phosphorus content in the crude oil, but also increase the content of diglyceride in the finished oil and increase the Crude oil yield and cost savings have brought huge benefits to oil companies. the

Detailed ways

In the following description, a lot of specific details are set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described here, and those skilled in the art can do it without departing from the meaning of the present invention. By analogy, the present invention is therefore not limited to the specific examples disclosed below. the

The experimental materials used in the following examples are as follows: expression vector pET-28a (+) and Escherichia coli JM109, BL21 (DE3) are preserved by our laboratory; Listeria monocytogenes (L.monocytogenes) Chinese industrial microorganism bacteria Species Collection and Management Center (CICC), the deposit number is: 21540, pMD18T-simple Vector, T4 DNA ligase and 10×T4 ligase Buffer were purchased from Bao Bioengineering (Dalian) Co., Ltd.; restriction enzymes Not I and EcoR I Purchased from BioLabs. the

The cloning of embodiment 1 phospholipase C gene

Primers were designed according to the sequence of the plc gene (YP005964174.1) of L.monocytogenes provided by NCBI, wherein the upstream primers were:

5'-CG GAATTC ATGTGTTGTGATGAATACTTACAAA-3' (the underlined part indicates the EcoR I restriction site);

Downstream primers are:

5'-AT GCGGCCGC TTATTCATTTGTTTTTTT-3' (the underlined part indicates the NotI restriction site).

Using the genomic DNA of Listeria monocytogenes (L.monocytogenes) with the preservation number CICC No. 21540 as a template, the lysophospholipase C gene was cloned by PCR, and connected with the vector pMD18T-simple to construct a clone Carrier pMD18T-simple-lm-plc, the cloning vector was transformed into Escherichia coli competent cells E.coli JM109, positive clones were selected and verified by sequencing. Cloning succeeded. the

Construction of embodiment 2 recombinant plasmid pET-L.m-plc

The recombinant vector pMD18T-simple-lm-plc and the expression vector pET-28a (+) were double digested with EcoR I and Not I, and the digestion reaction system was 20 μL: plasmid 10 μL, Buffer 32 μL, Not I0.5 μL, EcoR I0. 5 μL, 100*Bsa 0.2 μL, H2O 6.8 μL. The target gene and carrier DNA were recovered and ligated with T4 DNA ligase. The ligation reaction system was 10 μL: 6 μL of target gene, 1.2 μL of carrier DNA, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and 0.8 μL of H200, and reacted at 16°C for 12 hours. the

Transform the ligation product into Escherichia coli competent cells E.coli JM109, the transformation method is as follows:

(1) Add 10ul of the above ligation product to the E.coli JM109 competent cells (100ul/tube) that have been aliquoted, mix gently, and ice-bath for 30min;

(2) Put the above-mentioned EP tube in a water bath at 42°C, accurately time it for 90 seconds, and do not shake the EP tube;

(3) Transfer the EP tube to an ice bath and cool for 3 minutes;

(4) Add 890 μL of sterile TB medium to each tube, place on a shaker at 37°C, 100r/min, and recover for 1h;

(5) Centrifuge at 8000r/min for 2min, suck off 800μL of supernatant medium, gently blow and suck the remaining medium and cells with a pipette gun, and transfer to LB solid containing kanamycin at a final concentration of 100μg/ml Spread evenly on a plate and incubate in a 37°C incubator for 10-18 hours;

(6) Pick positive clones and verify the recombinant plasmid pET-L.m-plc by Not I and EcoR I double enzyme digestion. the

(7) Mix the successfully constructed recombinant plasmid pET-L.m-plc with an equal volume of 30% glycerol and store at -80°C. the

Example 3 Construction of recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC, deposit number CCTCC No.M2013301

Transform the constructed recombinant plasmid pET-L.m-plc into Escherichia coli BL21 (DE3), the transformation method is as follows: 

(1) Add 0.5ul of the above-mentioned recombinant plasmid pET-lm-plc to 100μL competent cells (100ul/tube) of E.coli BL21 (DE3) that has been aliquoted, mix gently, and ice-bath for 30min;

(2) Put the above-mentioned EP tube in a water bath at 42°C, accurately time it for 90 seconds, and do not shake the EP tube;

(3) Transfer the EP tube to an ice bath and cool for 3 minutes;

(4) Add 890 μL of sterile TB medium to each tube, place on a shaker at 37°C, 100r/min, and recover for 1h;

(5) Centrifuge at 8000r/min for 2min, suck off 800μL of supernatant medium, gently blow and suck the remaining medium and cells with a pipette gun, and transfer to LB solid containing kanamycin at a final concentration of 100μg/ml Spread evenly on a plate and incubate in a 37°C incubator for 10-18 hours;

(6) Pick the positive clones and verify the recombinant E. coli BL21(DE3)-pET28a-L.m-PLC by Not I and EcoR I double enzyme digestion. the

(7) Mix the successfully constructed recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC with an equal volume of 30% glycerol and store at -80°C. the

Example 4 Preparation of Phospholipase C by Recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC

Phospholipase C was prepared by recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC with the preservation number CCTCC No.M2013301. the

(1) Seed culture: Take 100 μL of the above-mentioned preserved recombinant E. coli BL21(DE3)-pET28a-Lm-PLC, CCTCC No.M2013301 and inoculate it into the seed medium, shake and culture it on a rotary shaker at 37°C for 10 h, shake the The rotation speed is 200r/min; the ingredients of the above seed medium are calculated in grams/liter: 10g of peptone, 5g of yeast powder, 10g of NaCl, and the rest is water. Sterilize at 1×10 ⁵ Pa for 20 minutes.

(2) Liquid fermentation culture: Inoculate the inoculum of the activated seed solution obtained through step (1) into the fermentation medium with an inoculum volume of 5% by volume, and the fermentation medium is filled in a 250ml Erlenmeyer flask with a liquid volume of 50ml. Shake culture at 35°C for 4 hours on a shaker with a shaker speed of 200r/min; add lactose to a final concentration of 2.5g/L, induce culture on a rotary shaker at 30°C for 20h with a shaker speed of 200r/min; ferment After completion, the fermentation broth is obtained; the above-mentioned fermentation medium components are calculated in grams/liter: peptone 10g, yeast powder 5g, glycerin 2g, and the remaining ingredients are 0.25mol/L Tris-HCl (pH7.2), and the fermentation medium is 1 × 105Pa under high pressure Sterilize for 20 minutes. the

(3) Extraction of crude enzyme liquid: Centrifuge the fermentation liquid obtained in step (2) at 4°C for 10 min at a speed of 10,000 r/min; collect the bacterial cell pellet and resuspend the bacterial cell with 25 mmol/L Tris-HCl (pH 7.2) , placed in a sonicator for 10 minutes of sonication, centrifuged the resulting sonicated solution, and took the supernatant, which was the intracellular crude enzyme solution. The enzyme activity of the intracellular crude enzyme liquid reached 376.026U/ml quantitatively measured by p-NPPC method. the

Example 5 Preparation of Phospholipase C by Recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC

(1) Seed culture: Take 100 μL of the above-mentioned preserved recombinant E. coli BL21(DE3)-pET28a-Lm-PLC, CCTCC No.M2013301 and inoculate it into the seed culture medium, shake and cultivate it on a rotary shaker at 32°C for 14 hours, shaker The rotation speed is 200r/min; the ingredients of the above seed medium are calculated in g/L: 10g of peptone, 6g of yeast powder, 10g of NaCl, and the rest of the ingredients are water. Sterilize under high pressure of 1×10 ⁵ Pa for 20min.

(2) Liquid fermentation culture: Inoculate the inoculum of the activated seed solution obtained through step (1) into the fermentation medium with an inoculum volume of 5% by volume, and the fermentation medium is filled in a 250ml Erlenmeyer flask with a liquid volume of 50ml. Shake culture at 30°C for 8 hours on a shaker, the shaker speed is 150r/min; add lactose to a final concentration of 5g/L, induce culture on a rotary shaker at 28°C for 14h, the shaker speed is 220r/min; add Glycine to a final concentration of 1g/L, continued shaking induction culture under the same conditions for 10h; fermentation was completed, to obtain a fermented liquid; the above-mentioned fermentation medium components in grams per liter: peptone 9g, yeast powder 12g, glycerol 3g, the rest of the composition 0.25mol/L Tris-HCl (pH7.2), sterilized under 1×10 ⁵ Pa high pressure for 20min.

(3) Extraction of crude enzyme liquid: centrifuge the fermentation liquid obtained in step (2) at 4°C for 10 min at a speed of 10,000 r/min, and collect the supernatant, which is the extracellular crude enzyme liquid; LTris-HCl (pH 7.2) resuspended bacteria, placed in a sonicator for 10 minutes of sonication, centrifuged the resulting sonicated solution, and took the supernatant, which was the intracellular crude enzyme solution; quantitatively measured by p-NPPC method The total enzyme activity of intracellular and extracellular crude enzyme solution reached 503.821U/ml. the

Example 6

(1) Seed culture: Take 50 μL of the above-mentioned preserved recombinant Escherichia coli BL21(DE3)-pET28a-Lm-PLC, CCTCC M2013301 and inoculate it into the seed culture medium, shake and culture it on a rotary shaker at 35°C for 12 hours, and the shaker speed is 180r /min; the ingredients of the above seed medium in grams/liter are: peptone 11g, yeast powder 6g, NaCl 11g, and the rest is water, and sterilized under 1×10 ⁵ Pa high pressure for 20 minutes.

(2) Liquid fermentation culture: Inoculate the inoculum of the activated seed solution obtained through step (1) into the fermentation medium with an inoculum volume of 5% by volume, and the fermentation medium is filled in a 250ml Erlenmeyer flask with a liquid volume of 50ml. Shake culture at 37°C for 6 hours on a shaker, the shaker speed is 180r/min; add lactose to a final concentration of 1g/L, induce culture on a rotary shaker at 25°C for 20h, the shaker speed is 200r/min; add Glycine to a final concentration of 3g/L, continued shaking induction culture under the same conditions for 18h; after the fermentation was completed, a fermentation liquid was obtained; the above-mentioned fermentation medium components were calculated in grams/liter: 12g of peptone, 22g of yeast powder, 4g of glycerin, and the remaining ingredients 0.25mol/L Tris-HCl (pH7.2), sterilized under 1×10 ⁵ Pa high pressure for 20min.

(3) Extraction of crude enzyme liquid: same as Example 5

The total enzyme activity of intracellular and extracellular crude enzyme liquid reached 779.358U/ml quantitatively measured by p-NPPC method. the

The rapeseed crude oil degumming of embodiment 7 recombinant PLC

Heat the rapeseed crude oil to 80°C, add a citric acid solution with a mass concentration of 45% and a mass of 0.5% rapeseed crude oil, and carry out acid pretreatment for 20 minutes under the stirring condition of 500r/min. Cool the acid-pretreated oil sample to 47°C, add a certain amount of 4% NaOH solution, and mix well to adjust the pH to 5.2. Add distilled water with 1% oil weight and 500U/kg recombinant phospholipase C, mix evenly, and stir at 300r/min at 62°C for 1h to carry out the enzyme reaction. After the reaction, the temperature of the reaction system was raised to 90° C. to inactivate the enzyme for 10 minutes, and centrifuged at 8000 r/min for 10 minutes to complete the catalytic degumming of rapeseed crude oil by the recombinant phospholipase C. the

After the crude rapeseed oil is degummed in this embodiment, the phosphorus content of the degummed rapeseed oil is 4.503 mg/kg, which is about 97.55% lower than that before degumming. the

The rice bran crude oil degumming of embodiment 8 recombinant PLC

Heat the crude rice bran oil to 80°C, add a citric acid solution with a mass concentration of 50% and a mass of 0.45% of the crude rice bran oil, and carry out acid pretreatment for 20 minutes under the stirring condition of 500r/min. Cool the acid-pretreated oil sample to 47°C, add a certain amount of 4% NaOH solution, and mix well to adjust the pH to 5.0. Add distilled water with 1% oil weight and 1000U/kg recombinant phospholipase C, mix evenly, and stir at 300r/min at 60°C for 1h to carry out the enzyme reaction. After the reaction, the temperature of the reaction system was raised to 90° C. to inactivate the enzyme for 10 minutes, and centrifuged at 10,000 r/min for 10 minutes to complete the catalytic degumming of crude rice bran oil by recombinant phospholipase C. the

After the crude rice bran oil is degummed in this embodiment, the phosphorus content of the degummed rice bran oil is 50.191 mg/kg, which is about 83.63% lower than that before degumming. the

The crude soybean oil degumming of embodiment 9 recombinant PLC

Heat the crude soybean oil to 80°C, add a citric acid solution with a mass concentration of 45% and a mass of 0.5% soybean crude oil, and carry out acid pretreatment for 20 minutes under the stirring condition of 500r/min. Cool the acid-pretreated oil sample to 47°C, add a certain amount of 4% NaOH solution, and mix well to adjust the pH to 5.2. Add distilled water with 1% oil weight and 500U/kg recombinant phospholipase C, mix evenly, and stir at 300r/min at 62°C for 1.2h to carry out the enzyme reaction. After the reaction, the temperature of the reaction system was raised to 90° C. to inactivate the enzyme for 10 minutes, and centrifuged at 10,000 r/min for 10 minutes to complete the catalytic degumming of crude soybean oil by recombinant phospholipase C. the

After the crude soybean oil is degummed in this embodiment, the phosphorus content of the degummed soybean oil is 3.945 mg/kg, which is about 98.29% lower than that before degumming. the

The crude soybean oil degumming of embodiment 10 recombinant PLC

Heat the crude soybean oil to 80°C, add a citric acid solution with a mass concentration of 45% and a mass of 0.5% soybean crude oil, and carry out acid pretreatment for 20 minutes under the stirring condition of 500r/min. Cool the acid-pretreated oil sample to 47°C, add a certain amount of 4% NaOH solution, and mix well to adjust the pH to 4.5. Add distilled water with 1% oil weight and 600U/kg recombinant phospholipase C, mix evenly, and stir at 300r/min at 67°C for 1h to carry out the enzyme reaction. After the reaction, the temperature of the reaction system was raised to 90°C to inactivate the enzyme for 10 minutes, and centrifuged at 10,000 r/min for 10 minutes to complete the catalytic degumming of crude soybean oil by recombinant phospholipase C. the

After the crude soybean oil is degummed in this embodiment, the phosphorus content of the degummed soybean oil is 3.705 mg/kg, which is about 98.40% lower than before degumming, and the diglyceride content in soybean oil is about 0.8% higher than before degumming . the

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention. the

Claims (10)

1. A recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC, the preservation number is CCTCC No.M2013301. 2. according to the described recombinant E. coli BL21(DE3)-pET28a-L.m-PLC of claim 1, CCTCCNo.M2013301, it is characterized in that: be made by following method: (1) Using the genome of Listeria monocytogenes (L.monocytogenes) with the preservation number CICC No. 21540 as a template, the phospholipase C gene was cloned, and its base sequence is shown in SEQ ID NO: 1; (2) Cloning the phospholipase C gene obtained in step (1) into the expression vector pET-28a(+) to obtain a recombinant vector; (3) Transform Escherichia coli competent cells with the recombinant vector obtained in step (2) to construct recombinant Escherichia coli. 3. The method for preparing phospholipase C by using the recombinant Escherichia coli BL21 (DE3)/pET-lm-plc, CCTCCNo.M2013301 according to claim 1 or 2, characterized in that: the recombinant Escherichia coli is used as a fermentation strain for liquid fermentation, Preparation of Phospholipase C. 4. the method for preparing phospholipase C according to claim 3 is characterized in that: comprising, (1) Seed culture: inoculate the recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC, CCTCCNo.M2013301 in the seed medium, shake and culture at 28°C-37°C for 6h-14h, shaker speed 150- 220r/min; (2) Liquid fermentation culture: inoculate the fermentation medium with the inoculum amount of 1-10% by volume of the activated seed liquid obtained through the cultivation in step (1), shake and culture at 28-37°C for 4h-6h, and shaker speed 150 ~220r/min; then add lactose, shake induction culture at 18℃~32℃ for 10~25h, shaker speed 150~220r/min; finally add glycine, continue shaking induction culture under the same conditions for 20~40h, the fermentation is complete, to obtain a fermented liquid; (3) Extraction of crude enzyme solution: Centrifuge the fermented solution obtained in step (2); collect the supernatant, which is the extracellular crude enzyme solution; collect the bacterial cell pellet and resuspend it with Tris-HCl, then ultrasonically break it, and ultrasonicate the obtained Centrifuge the crushed solution, collect the supernatant, which is the intracellular crude enzyme solution. 5. The method for preparing phospholipase C according to claim 4, characterized in that: the components of the seed medium in step (1) are calculated in grams per liter: peptone 8-12g, yeast powder 3-10g, NaCl 8-12g , the remaining ingredients are water, pH7.2～7.6. 6. The method for preparing phospholipase C according to claim 4, characterized in that: the components of the fermentation medium in step (2) are calculated in grams per liter: peptone 8-12g, yeast powder 5-25g, glycerin 0-25g 6g, and the remaining components are 0.25mol/L Tris-HCl (pH7.2). 7. The method for preparing phospholipase C according to claim 4, characterized in that: the amount of lactose added in step (2) is 1-10 g per liter of the fermentation medium. 8 . The method for preparing phospholipase C according to claim 4 , characterized in that: the amount of glycine added in step (2) is 0-5 g per liter of the fermentation medium. 9. A method for vegetable crude oil degumming, characterized in that: comprising, Preheating and acid pretreatment of vegetable crude oil; Add phospholipase C as claimed in claim 3 and react; Inactivate the enzyme and centrifuge to complete the degumming. 10. according to the method for vegetable crude oil degumming described in claim 9, it is characterized in that: concrete steps are as follows: (1) Heat vegetable crude oil in a water bath in a stoppered Erlenmeyer flask to 60-80°C, add a citric acid solution with a mass concentration of 0.5% of the total crude oil mass and a mass concentration of 25%-50%, and stir at 300-500r/min 15-25 minutes of acid pretreatment under the conditions. (2) Cool the acid-pretreated oil sample to 40-60°C, add a certain amount of 4% NaOH solution and mix well to adjust the pH to 4-7. (3) Add distilled water with an oil weight of 1% to 3%, and the phospholipase C at 400 to 2000 U/Kg, mix evenly, and stir at 300r/min to 500r/min at 40°C to 75°C for 1 to 2 hours, Perform an enzymatic reaction. (4) Elevate the temperature of the reaction system to above 90° C. to inactivate the enzyme for 10 minutes, and perform centrifugation at 8,000 to 10,000 r/min for 10 minutes to complete the catalyzed degumming of vegetable crude oil by the phospholipase C.