Phosphatidase C mutant and use thereof

A technology of phospholipase and application, which is applied in the fields of application, hydrolase, and fat production, and can solve the problems of less research on heterologous expression

Active Publication Date: 2015-05-20
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few studies on the heterologous expression of BC-PC-PLC, and there have been reports involving the expression of BC-PC-PLC in Bacillus subtilis (Bacillus subtilis) and Pichia pastoris (pichia pastoris) (see, for example Durban, M.A., Silbersack, J., Schweder, T., Schauer, F., Bornscheuer, U.T. (2007) High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis. Appl Microbiol Biotechnol 74(3):634-639; and Seo, K.H, Rhee J.I.(2004)High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization.Biotechnol Lett26(19):1475-1479)

Method used

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  • Phosphatidase C mutant and use thereof
  • Phosphatidase C mutant and use thereof
  • Phosphatidase C mutant and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: Construction of wild-type BC-PC-PLC Pichia pastoris expression strain

[0099] According to the mature peptide sequence (PDB ID: 1AH7) of the phosphatidylcholine-specific phospholipase C of Bacillus cereus and the codon preference of Pichia pastoris, the DNA sequence of BC-PC-PLC (SEQ ID No: 1) was designed ), and at its front fusion α factor signal peptide sequence (its DNA sequence is derived from commercial Pichia pastoris expression vector pPIC-9k, the 8th-274th in SEQ ID No:3) and the Kozak sequence of Pichia pastoris (SEQ 1-7 in ID No:3), and finally obtained the α-BC-PC-PLC DNA sequence (SEQ ID No:3).

[0100] The α-BC-PC-PLC DNA sequence was provided to Shanghai Sangon Biotechnology Co., Ltd. for whole gene synthesis, and the cloning vector pGEM-T-PLC containing the α-BC-PC-PLC DNA sequence was obtained. Using this vector as a template, use HS DNA polymerase and primer pair AmPLC-3 / AmPLC-4 were used to amplify the PLC fragment by PCR.

[0101] Usi...

Embodiment 2

[0105] Example 2: Construction and screening of BC-PC-PLC mutant library

[0106] Using the pAO-PLC vector as a template, use HS DNA polymerase and primer pair AmPLC-1 / AOXH-2, a fragment of about 900bp was amplified by PCR. Using the pAO-PLC vector as a template, use HS DNA polymerase and primer pair AOXH-3 / AmPLC-4, amplify about 1.1kb fragment by PCR, mix about 900bp fragment and about 1.1kb fragment obtained by the previous two steps of PCR as the template of the third step PCR, use the primer pair AmPLC-1 / AmPLC-4 and HS DNA polymerase, a fragment of about 1.9kb was amplified by PCR.

[0107] The about 1.9kb fragment was cloned into pAO-PLC through AatII and EcoRI restriction sites to obtain pmAO-PLC. In pmAO-PLC, a HindIII restriction site in pAO-PLC was mutated so that only a HindIII restriction site located at the 5' end of the BC-PC-PLC sequence was retained, so that BC could be converted to BC using HindIII and EcoRI - The mutated fragment of PC-PLC was cloned i...

Embodiment 3

[0110] Embodiment 3: BC-PC-PLC mutant sequence analysis

[0111] The 7-3-3 strain was inoculated in 3ml YPD liquid medium, cultivated overnight at 30°C, and the genomic DNA was extracted. Using the genomic DNA of the 7-3-3 strain as a template, use HS DNA polymerase and primers were used for PCR amplification of AOX1-5 / AOX1-3 to obtain the DNA sequence of BC-PC-PLC in the 7-3-3 strain. The obtained sequence was sent to Shanghai Sangon Bioengineering Co., Ltd., and AOX1-5 / AOX1-3 was sequenced with primers. The DNA sequencing result of 7-3-3 BC-PC-PLC is shown in SEQ ID No:4. After comparison, it was found that compared with SEQ ID No: 3, there were 7 base mutations in SEQ ID No: 4, including three sense mutations, which were the mutation of G at position 59 to A, making the Arginine at position 20 is mutated to histidine (CGT→CAT); A at position 188 is mutated to G, asparagine at position 63 is mutated to serine (AAC→AGC); C at position 248 is mutated to A, making Alanine ...

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Abstract

The present application provides a wild type phosphatidylcholine specificity phospholipase C (PLC) mutant of Bacillus cereus, the related mutations comprising asparagine at position 63 mutating to another amino acid; also comprising mutation of arginine to histidine at position 20 and of alanine to aspartic acid at position 83. The present application also provides a nucleic acid molecule encoding said mutant, a vector containing said nucleic acid molecule, and a cell containing said nucleic acid molecule or vector. The present application also provides uses of said mutant, nucleic acid molecule vector, and cell.

Description

field of invention [0001] The present application relates to phosphatidylcholine-specific phospholipase C mutants and uses thereof. Background of the invention [0002] Degumming is an important step in oil refining. The traditional hydration degumming method has high economic costs, high material energy consumption, and heavy environmental pollution. Therefore, in recent years, a lot of work has been devoted to using enzymatic degumming in the degumming link of oil refining. Compared with traditional methods, enzymatic degumming can improve economic benefits, realize energy saving and emission reduction, and cause less pollution to the ecological environment. It has great advantages in terms of environmental protection, economy, and quality. One enzyme used in oil degumming is phospholipase. Compared with other degumming enzymes, phospholipase C (PLC) showed greater advantages, for example, increasing the yield of diacylglycerol (DAG) and reducing the loss of oil yield. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/81C12N1/19C11B3/00C12R1/085C12R1/84
Inventor 许骏宣姚吉李金敏
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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