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Novel phosphatidase B and application thereof

A technology of phospholipase and application, which is applied in the field of genetic engineering of enzymes, and can solve problems such as difficult to obtain satisfactory results

Active Publication Date: 2017-05-17
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is generally difficult to obtain satisfactory results for a gene mutated once, so a sequential error-prone PCR (Sequential Error-prone PCR) strategy has been developed.

Method used

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  • Novel phosphatidase B and application thereof
  • Novel phosphatidase B and application thereof
  • Novel phosphatidase B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Obtaining of wild-type phospholipase B gene

[0053] 1. The wild-type phospholipase B gene plb is from Saccharomyces cerevisiae TCCC31028, and its genomic DNA is extracted, wherein the extraction steps of Saccharomyces cerevisiae genomic DNA are as follows:

[0054] (1) Pick a ring of bacteria from the culture plate and inoculate in 50mL of appropriate medium, culture at 30°C and 250r / min for 16-18h.

[0055] (2) Afterwards, take 1 mL of the culture solution in a 1.5 mL EP tube, centrifuge at 12,000 r / min for 2 min, pour off the supernatant, resuspend with 200 μL of lysate, add quartz sand, and shake for 20-30 min.

[0056] (3) Add 500uL of lysate, centrifuge at 12000r / min for 5min, and take the supernatant.

[0057] (4) Add an equal volume of Tris to balance phenol:chloroform=1:1, mix well, centrifuge at 12000rpm for 5min, and transfer the supernatant to another EP tube.

[0058] (5) Repeat the extraction twice until no protein layer appears, and finall...

Embodiment 2

[0075] Embodiment 2: the acquisition of phospholipase B mutant gene plbm

[0076] 1. Random mutation based on error-prone PCR technology

[0077] In the error-prone PCR reaction system, P1 and P2 were used as upstream and downstream primers, and plb was used as a template to perform error-prone PCR to obtain plbm.

[0078] The reaction conditions for its amplification are:

[0079]

[0080]

[0081] The amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 70°C for 40 s, extension at 72°C for 2 min, and 30 cycles of reaction; extension at 72°C for 10 min.

[0082] 2. The phospholipase B error-prone PCR product is cloned into the expression vector pET28a, transformed into Escherichia coli BL21, inoculated in a 96-well cell culture plate containing 200 μL of LB liquid medium (containing 30 μg / mL of Kan) in each well, at 37 Cultivate on a shaker at 200r / min under the condition of ℃, when the OD600 reaches 0.6, add I...

Embodiment 3

[0101] Example 3: Construction of Pichia pastoris free expression recombinant bacteria

[0102] 1. Construction of wild-type phospholipase B and phospholipase B mutant expression vectors pPIC9K-plb and pPIC9K-plbm

[0103] plb and plbm were respectively digested with Pichia pastoris expression vector pPIC 9K by EcoRI and NotI, then ligated, transformed into Escherichia coli JM109 competent cells, and Amp was selected r The positive transformant, the plasmid was extracted after colony culture, and the enzyme digestion verification was successful (see figure 2 ) to obtain recombinant expression vectors pPIC9K-plb and pPIC9K-plbm.

[0104] 2. Construction of wild-type phospholipase B and phospholipase B mutant expression recombinant strains

[0105] (1) Linearization of plasmid DNA

[0106] Before transforming Pichia pastoris, the recombinant expression plasmids pPIC9K-plb and pPIC9K-plbm were linearized with SalI restriction endonuclease.

[0107] (2) Electroporation of lin...

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Abstract

The invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to a method for obtaining phosphatidase B mutants with improved enzyme activity through a PCR (sequential error-prone) technology in vitro directed evolution, using the phosphatidase B mutants to prepare glycerophosphatidylcholine and performing oil degumming. The phosphatidase B mutant PLBM is obtained; the enzyme activity is improved by 25 percent through being compared with that of wild phosphatidase B.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of genetic engineering of enzymes, and in particular relates to a phospholipase B mutant with improved enzyme activity obtained through in vitro directed evolution by error-prone PCR technology, and a method for preparing glycerol phosphatidylcholine and oil degumming by using the same. [0003] Background technique: [0004] Phospholipase B (PLB) is widely distributed in animals, plants and microorganisms, and has the activities of hydrolase and lysophospholipase-transacylase. Hydrolase activity removes fatty acids from phospholipids and lysophospholipids, while transacylase activity transfers free fatty acids to lysophospholipids to generate phospholipids. [0005] Glycerol phosphatidylcholine (L-alpha glycerylphosphorylcholine, L-α-GPC) is composed of choline, glycerol and phosphate, and is the precursor for the synthesis of acetylcholine neurotransmitter. It has important medical application valu...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/81C12N1/19C12P13/00C11B3/00C12R1/84
CPCC11B3/003C12N9/18C12N15/815C12N2800/102C12P13/001C12Y301/01005
Inventor 刘逸寒路福平李明杰
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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