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Recombinant escherichia coli, method for preparing phospholipase C and method for degumming soybean crude oil

A technology of recombinant Escherichia coli and phospholipase, which is applied in the fields of biotechnology and oil degumming, can solve problems such as unsatisfactory acid and heat tolerance, limited application, and safety, and achieve low cost, convenient enzyme source, and reduced loss Effect

Inactive Publication Date: 2014-01-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at the same time, there are some unavoidable problems, such as: most of the original strains producing PLC are pathogenic bacteria, and there are potential safety hazards in food application; compared with traditional acid degumming, enzymatic degumming still faces the problem of higher cost , if the effective immobilization of PLC can be achieved, the cost of oil refining can be greatly reduced; in addition, PLC from common microorganisms has poor tolerance to acid and heat, which greatly limits its application in the food industry, so it needs to be screened Or modify the PLC with stronger acid resistance and heat resistance

Method used

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  • Recombinant escherichia coli, method for preparing phospholipase C and method for degumming soybean crude oil
  • Recombinant escherichia coli, method for preparing phospholipase C and method for degumming soybean crude oil

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The composition of the seed medium is: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, pH 7.4, sterilized at 121°C for 20min.

[0034] The components of the fermentation medium are: peptone 12g / L, yeast extract 24g / L, glycerol 0.4% (v / v), KH 2 PO 4 170mM, K 2 HPO 4 720mM, 121°C, sterilized for 20min.

[0035] Inoculate the recombinant Escherichia coli BL21(DE3)-pET28a-Sa-PLC in the seed medium containing kanamycin sulfate 50mg / mL, and culture it in a shake flask at 37°C and 200r / min until the logarithmic growth phase, as the seed solution Then inoculate the seed liquid into 20mL fermentation medium containing kanamycin sulfate 50mg / mL by 5% inoculum size, and culture it in 200r / min shake flask at 37°C until OD 600 =0.6, then add IPTG to a final concentration of 0.05mM, induce culture at 30°C, 200r / min for 8h; centrifuge the fermentation broth at 4°C, 8000r / min for 10min, collect the bacteria, and use 50mM Tris- Resuspend the bacteria in HCl, sonicate for 10 minutes,...

Embodiment 2

[0037] The composition of the seed medium is: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, pH 7.4, sterilized at 121°C for 20min.

[0038] The components of the fermentation medium are: peptone 12g / L, yeast extract 24g / L, glycerol 0.4% (v / v), KH 2 PO 4 170mM, K 2 HPO 4 720mM, 121°C, sterilized for 20min.

[0039] Inoculate the recombinant Escherichia coli BL21(DE3)-pET28a-Sa-PLC in the seed medium containing kanamycin sulfate 50mg / mL, and culture it in the shake flask at 37°C and 180r / min until the logarithmic growth phase, as the seed solution ;Then inoculate the seed liquid into 20mL fermentation medium containing kanamycin sulfate 50mg / mL by 1% inoculum size, and culture it in shake flask at 37°C and 180r / min until OD 600 =1.0, then add IPTG to a final concentration of 0.005mM, induce culture at 37°C, 180r / min for 5h; centrifuge the fermentation broth at 4°C, 8000r / min for 10min, collect the bacteria, and use 50mM Tris- The cells were resuspended in HCl, sonicated fo...

Embodiment 3

[0041] The composition of the seed medium is: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, pH 7.4, sterilized at 121°C for 20min.

[0042] The components of the fermentation medium were: peptone 12g / L, yeast extract 24g / L, glycerin 0.4% (v / v), sterilized at 121°C for 20min.

[0043] Inoculate the recombinant Escherichia coli BL21(DE3)-pET28a-Sa-PLC in the seed medium containing kanamycin sulfate 50mg / mL, and culture it in a shake flask at 37°C and 200r / min until the logarithmic growth phase, as the seed solution ;Then inoculate the seed liquid into 20mL fermentation medium containing kanamycin sulfate 50mg / mL according to the inoculation amount of 3%, directly add IPTG to the final concentration of 0.001mM, and induce culture at 30°C and 180r / min for 8h ;Centrifuge the fermentation broth at 4°C, 8000r / min for 10min, collect the cells, resuspend the cells with Tris-HCl of pH 7.2, ultrasonically break for 15min, and centrifuge the cell debris at 4°C, 10000r / min for 10min, The...

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Abstract

The invention discloses recombinant escherichia coli, a method for preparing phospholipase C and a method for degumming soybean crude oil. The method for preparing phospholipase C (PLC) is finished by the following steps: (1) cloning by taking genome deoxyribonucleic acid (DNA) of staphylococcus aureus of which the preservation number is GIM1.142 (ATCC6538) as a template to obtain a phospholipase C gene, wherein a base sequence of phospholipase C gene is shown in SEQ ID NO:1 (2) cloning the phospholipase C gene obtained in the step (1) to a pET-28a(+) expression carrier, and building recombinant expression plasmids; (3) transforming the recombinant expression plasmids obtained in the step (2) into escherichia coli BL21(DE3) competent cells, so as to obtain the recombinant escherichia coli BL21(28a-S.a PLC). In the method for degumming soybean crude oil, phospholipase C produced by the recombinant escherichia coli is taken as a catalyst, so that the enzyme source is convenient, the yield is high, the fermentation technology is simple, and the cost is low. Compared with the traditional enzyme degumming, the method for degumming soybean crude oil by adopting phospholipase C has the advantages that the reaction time can be shortened; loss of neutral oil is reduced; the refining yield is improved. Therefore, the degumming method disclosed by invention has a certain application prospect in the field of the oil production industry.

Description

technical field [0001] The invention relates to the fields of biotechnology and oil degumming, in particular to a method for preparing phospholipase C by a strain of recombinant Escherichia coli and a method for degumming soybean crude oil catalyzed by the enzyme. Background technique [0002] Phospholipase C (PLC) can catalyze the hydrolysis of the Sn-3 ester bond of phospholipids to produce diacylglycerol (DAG) and phosphorylated head groups. According to the substrate specificity of PLC, PLC derived from bacteria can be mainly divided into two categories: phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-preferring phospholipase C (PC-PLC). . The hydrolysis products of PLC inositol triphosphate (IP3) and diglyceride (DAG) play an important role in information transmission and cell metabolism, and the former can promote Ca 2+ Released from the calcium pool; DAG can activate protein kinase K (PKC), thereby triggering a series of intracellular ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/16C12N15/55C11B3/00C11B3/04C12R1/19
Inventor 常明芮丽莲刘睿杰金青哲王兴国刘元法
Owner JIANGNAN UNIV
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