Phospholipase and application thereof

A technology of phospholipids and polynucleotides, applied in the field of phospholipases, can solve problems such as ineffectiveness

Active Publication Date: 2018-06-29
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

G999 is a lysophospholipase A that acts only on lysophospholipids, PLC is an artificially modified phospholipase C that can rapidly act on substrates such as phosphatidylcholine and phosphatidylethanolamine, but hardly acts on phosphatidylinositol and phosphatidic acid

Method used

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  • Phospholipase and application thereof
  • Phospholipase and application thereof
  • Phospholipase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1: phospholipase error-prone PCR of the present invention

[0120] EPCR system (50ul):

[0121]

[0122] Using SEQ ID NO: 1 as a template, using the sequence shown in SEQ ID NO: 3 as an upstream primer and the sequence shown in SEQ ID NO: 4 as a downstream primer to perform error-prone PCR.

Embodiment 2

[0123] Embodiment 2: Construction of phospholipase mutant library of the present invention

[0124] Use 1% agarose (purchased from AMRECSO) gel electrophoresis to separate and purify the error-prone PCR product target band, and use the E.Z.N.ATM gel recovery kit from OmegaBio-Tek, USA to recover the target band. The products recovered from the gel were digested with two restriction endonucleases AvrII and NotI-HF, and then the digested products were recovered with the E.Z.N.ATM product recovery kit from OmegaBio-Tek Company. Simultaneously carry out enzyme digestion of pPIC9K vector and recovery of enzyme digestion products.

[0125] Take an appropriate amount of the above two recovered enzyme digestion products, mix them, add a certain amount of ligase and ligase buffer, and place them in a water bath at 22°C for about 2 hours to react.

[0126] Take out one tube containing 100 μl of DH5α competent cells, after the ice bath melts, add 20 μl of the ligation product accordingl...

Embodiment 3

[0132] Example 3: Plate Screening of Phospholipase Mutant Library

[0133] Plate all the bacteria of ECSPLB-pPIC9K-GS115 mutant onto BMMY plate (1% yeast extract, 2% peptone, 100mM potassium phosphate pH6.0, 1.34% YNB 4×10-5% biotin, 0.5% methanol, 1 % soybean lecithin, 1.5% agar), cultured at 30°C for 3 days, the results were as follows figure 1 shown. figure 1 Among the four plates, one bacterium in the middle of each plate is the phospholipase parent, and the other bacteria are phospholipase variants. Pick forward phospholipase variants with larger clear and cloudy circles than the parental circle. figure 1 In the middle, the black square marked in the upper left picture is the bacteria containing phospholipase variant 154, the black square marked in the upper right picture is the bacteria containing phospholipase variant 2, and the black square marked in the lower left picture is the bacteria containing phospholipase variant 10 Bacteria containing phospholipase variant ...

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Abstract

The invention relates to phospholipase and an application thereof. Specifically, the invention provides a polypeptide selected from: (1) a polypeptide having substitution mutations at at least one ofpositions 182, 221, 274, 323, 339, 342, 369 and 500 of an amino acid sequence shown in SEQ ID NO: 2; (2) a polypeptide comprising the polypeptide shown in (1) and a polypeptide that promotes expression and purification of the polypeptide shown in (1). The invention further provides a polynucleotide sequence encoding the polypeptide, a nucleic acid construct containing the polynucleotide sequence,a host cell, and related uses. The polypeptide provided by the invention is phospholipase and can be used in oil refining, phospholipid modification, feed modifiers, food industry and medicine industry.

Description

technical field [0001] The present invention relates to phospholipase and its application. Background technique [0002] Phospholipase (PL) is an enzyme that can hydrolyze glycerophospholipids in living organisms. According to the site of phospholipase hydrolyzing glycerophospholipids (Richmond G.S. et al., Int.J.Mol.Sci., 2011, 12:588-612), Phospholipases can be classified into phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase B (PLB), phospholipase C (PLC), phospholipase D (PLD) and lysophospholipase A. [0003] PLA1 can hydrolyze the Sn-1 acyl ester bond of difatty acylphospholipids to produce lysophospholipids and fatty acids. PLA2 can hydrolyze the acyl ester bond at the Sn-2 position to produce lysophospholipids and fatty acids. PLB is a phospholipase that can hydrolyze the two acyl ester bonds at the Sn-1 and Sn-2 positions of di-fatty acyl phospholipids to produce glycerol phosphorylcholine and free fatty acids. The enzyme also has lysophospholipase a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63C11B3/00C11C3/00A23K20/147A23K20/153A23L33/13A23L33/18C11D3/386
CPCC11B3/00C11B3/003C11C3/00C11D3/38636C12N9/18C12Y301/01005A23V2002/00A23V2250/55
Inventor 周美凤徐正军戴小军牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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