Efficient zinc ion independent phospholipase C mutant

A technology of phospholipase and lysine, applied in the field of phospholipase C mutants, can solve the problems of shortened shelf life, poor color and taste, and affecting refining effect.

Inactive Publication Date: 2017-06-23
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, phospholipids will cause color and taste to deteriorate, shorten the shelf life and affect the subsequent refining effect

Method used

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  • Efficient zinc ion independent phospholipase C mutant
  • Efficient zinc ion independent phospholipase C mutant
  • Efficient zinc ion independent phospholipase C mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142]Example 1: PLC-N63DN131SN134D mutant library construction and screening

[0143] 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: 8, The amino acid sequence encoded by it is shown in SEQ ID NO: 9), and the α factor signal peptide sequence and the Kozak sequence of Pichia pastoris are fused at its front end to finally obtain the α-BC-PC-PLC DNA sequence (SEQ ID NO: 10 ).

[0144] 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 The DNA polymerase and the primer pair AmPLC-3 / AmPLC-4 are used to amplify the PLC fragment by PCR.

[0145] Using the pPIC-9k expression vector as a template, use DNA polymerase and primer pair A...

Embodiment 2

[0154] Embodiment 2: PLC-N63DN131SN134D mutant sequence analysis

[0155] Inoculate 31# strain in 3ml YPD liquid medium, culture overnight at 30°C, and extract genomic DNA. With the genomic DNA of the 31# strain as a template, use AOX1-5 / AOX1-3 was amplified by DNA polymerase and primers to obtain the DNA sequence of PLC in 31# strain. The obtained sequence was sent to Shanghai Sangon Bioengineering Co., Ltd., and AOX1-5 / AOX1-3 was sequenced with primers. The DNA sequencing results of 31# PLC had 2 base mutations, the 56th tyrosine was mutated to histidine (TAT→CAT); the 106th methionine was mutated to valine (ATG→GTG). The sequence is shown in SEQ ID NO:3.

Embodiment 3

[0156] Example 3: Construction and screening of PLC-N63DN131SN134D mutant single point mutant Pichia pastoris expression strain

[0157] 1. Construction and screening of PLC-N63DN131SN134D-Y56H

[0158] Using the pmAO-PLC-N63DN131SN134D vector as a template, use DNA polymerase and primer pair EPPLC-1 / 56H-2, a fragment of about 180bp was amplified by PCR. Using the pmAO-PLC-N63DN131SN134D vector as a template, use DNA polymerase and primer pair 56H-3 / EPPLC-2 were used to amplify about 570bp fragment by PCR. The about 180bp fragment and the about 570bp fragment obtained in the previous two steps of PCR were mixed as the template of the third step PCR, and the primer pair EPPLC-1 / EPPLC-2 and DNA polymerase, a fragment of about 755bp was amplified by PCR.

[0159] The about 755bp fragment was cloned into pmAO-PLC through HindIII and EcoRI restriction sites to obtain pmAO-PLC-N63DN131SN134D-Y56H vector. pmAO-PLC-N63DN131SN134D-Y56H was linearized with SalI, and the 8.5kb fr...

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Abstract

The invention relates to an efficient zinc ion independent phospholipase C mutant. Specifically, the invention provides a separated amino acid sequence, wherein the amino acid sequence includes (1) an amino acid sequence shown as SEQ ID NO:7, or (2) polypeptide which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (1), reserves phospholipase C activity of the SEQ ID NO:7 and is derived from (1). The invention also relates to a polynucleotide sequence for coding the amino acid sequence, a nucleic acid construct and a host cell containing the polynucleotide sequence, a composition containing the amino acid sequence and an application of the amino acid sequence.

Description

technical field [0001] The invention relates to an efficient zinc ion-independent phospholipase C mutant, in particular to a phosphatidylcholine-specific phospholipase C mutant obtained through a mutation screening method in molecular biology and its application. Background technique [0002] Degumming is an important step in oil refining, but the traditional hydration degumming method has high economic costs, high material energy consumption, and heavy environmental pollution. Therefore, in recent years, many efforts have been devoted to using enzymatic degumming in the degumming link of oil refining. , and made great progress. Compared with traditional methods, enzymatic degumming can improve economic benefits, achieve energy saving and emission reduction, and cause less pollution to the ecological environment. It has great advantages in environmental protection, economy, and quality. The enzyme used in oil degumming is phospholipase. Phospholipases have the ability to h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C11B3/00
CPCC11B3/003C12N9/20C12Y301/04003C11B3/00C12N15/63C12N9/16C12R2001/085C12N1/205C12P7/00
Inventor 宣姚吉刘莎顾思天包悦佚牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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