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Phosphatidase, coding gene, preparation method and application thereof

A phospholipase and coding technology, applied in the fields of coding genes, phospholipase, and preparation, can solve the problems of low temperature stability, affecting the quality of finished products, temperature stability of residual phospholipase, etc., achieving broad pH stability, broad application prospects, The effect of excellent thermal stability

Active Publication Date: 2017-05-24
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] In summary, the existing commercial phospholipases and existing reported phospholipases still cannot meet the needs of practical applications.
For example, in the enzymatic degumming process of vegetable oil, some commercial phospholipases have a high pH value, residual lipase activity, and low temperature stability or high temperature stability of some phospholipases, resulting in residues that affect the later stage. Finished product quality, etc.

Method used

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  • Phosphatidase, coding gene, preparation method and application thereof
  • Phosphatidase, coding gene, preparation method and application thereof
  • Phosphatidase, coding gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1: Separation and purification of wild polypeptide enzyme of the present invention and identification of partial amino acid sequence information

[0138]Take 2 freshly cultured Cladosporium WBRD00050 (CGMCC No.7508) slopes (incubate on PDA medium at 28°C for 120 h), add 10 ml of sterile water to each slope, wash thoroughly to obtain a spore suspension, and combine all the spore suspensions liquid. Then inoculate the spore suspension into the fermentation shaker flask according to the inoculum amount of 1ml / bottle. The fermentation shaker flask has a specification of 250ml, and the filling volume is 50ml. The culture conditions of the fermentation shake flask are: 28° C., 200 rpm, and the culture time is 120 hours.

[0139] The composition of the fermentation medium is as follows:

[0140] Sucrose 1%, peptone 0.5%, yeast extract powder 0.5%, corn steep liquor 0.5%, Na 2 HPO 4 0.25%, KH 2 PO 4 0.15%, magnesium sulfate 0.1%, zinc sulfate heptahydrate 0.2%, a...

Embodiment 2

[0164] Embodiment 2: the cloning of polypeptide coding polynucleotide sequence of the present invention

[0165] The total RNA of Cladosporium WBRD00050 was extracted according to the instructions of Qiagen RNeasy Plant Mini Kit. According to the instructions of Promega Reverse Transcription Kit, RNA was reverse transcribed into cDNA. Then, degenerate primers were designed according to the amino acid sequence information of the three peptides obtained in Example 1. SEQ ID NO:3 is an upstream primer designed based on the sequence of PEAEY in peptide 1, and its degeneracy is 128. SEQ ID: 4 is an upstream primer designed based on the YQPVE sequence in peptide 2, and its degeneracy is 128. SEQ ID NO:5 is an upstream primer designed based on the GAGFD sequence in peptide 3, and its degeneracy is 256.

[0166] Using SEQ ID NO: 3-5 as an upstream primer, oligo dT as a downstream primer for PCR, and using the cDNA prepared above as a template to perform PCR. The PCR conditions are...

Embodiment 3

[0169] Embodiment 3: Construction of polypeptide expression vector and engineering bacteria of the present invention

[0170] SignalP 4.1server software (http: / / www.cbs.dtu.dk / services / SignalP / ) analyzed the polypeptide shown in SEQ ID NO: 2, and as a result, the amino acid sequence at positions 1-20 was determined to be the signal peptide sequence. Then, based on the sequence without the signal peptide, the primers were designed. The upstream primer is shown in SEQ ID NO: 8, and the downstream primer is SEQ ID NO: 9.

[0171] Using the cDNA prepared in Example 2 as a template, the upstream and downstream primers shown in SEQ ID NO:8 and SEQ ID NO:9 were used as primers to clone the target sequence.

[0172] Use 1% agarose gel electrophoresis to separate and purify the 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 Avr II and Not I, and ...

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Abstract

The invention provides phosphatidase, a coding gene, a preparation method and application thereof. Specifically, the invention provides isolated polypeptide, which contains: (1) the 21st to 565th amino acid sequence of SEQ ID NO:2; or (2) a polypeptide derived from (1) after substitution, deletion or addition of one or more amino acids in the amino acid sequence of (1) and reservation of the 21st to 565th amino acid sequence of SEQ ID NO:2. The invention also relates to a polynucleotide sequence for coding the polypeptide, a nucleic acid construct containing the polynucleotide sequence, a host cell of the polynucleotide sequence or the nucleic acid construct, a composition containing the polypeptide, and application of the polypeptide.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to phospholipase, coding gene, preparation method and application thereof. Background technique [0002] Phospholipase (Phospholipase, PL) is an enzyme that can hydrolyze glycerophospholipids that exists in organisms, depending on the site of phospholipase hydrolysis of glycerophospholipids (Richmond G.S. et al., Int.J.Mol.Sci.2011, 12:588-612) , phospholipases can be divided into phospholipase A1 (PLA 1 ), phospholipase A2 (PLA 2 ), phospholipase B (PLB), phospholipase C (PLC), phospholipase D (PLD) and lysophospholipase A (LysoPLA), as shown in the following formula: [0003] [0004] R in the formula 1 and R 2 is a fatty acyl group; R 3 For amino alcohols, such as choline, ethanolamine, inositol and serine. [0005] PLA 1 It can hydrolyze the Sn-1 acyl ester bond of difatty acyl phospholipids to produce lysophospholipids and fatty acids. PLA 2 It can hydrolyze ...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/63C11B3/00A23K1/165
Inventor 徐正军周美凤许骏杨天奎
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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