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Moniliophthora perniciosa laccase and engineering bacteria, recombinant laccase as well as application thereof

A technology of engineering bacteria and laccase, which is applied in the field of environmental biology and biology, can solve the problems of easy loss of activity, sensitivity to chloride ions, application limitations of fungal laccase, etc., and achieve the effect of good pH stability and high tolerance

Active Publication Date: 2015-08-05
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The research on fungal laccase is relatively in-depth, but its reaction pH is generally in the acidic range, it is easy to lose activity in alkaline environment, and it is relatively sensitive to chloride ions; this limits the application of fungal laccase in the industrial field.

Method used

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  • Moniliophthora perniciosa laccase and engineering bacteria, recombinant laccase as well as application thereof
  • Moniliophthora perniciosa laccase and engineering bacteria, recombinant laccase as well as application thereof
  • Moniliophthora perniciosa laccase and engineering bacteria, recombinant laccase as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Synthesis of Novel Laccase Gene

[0028] Based on the American National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ) GenBank accession number: AFD97050 amino acid sequence based on the codon preference of Pichia pastoris The novel laccase gene was optimized to obtain a new type of laccase gene, whose sequence is shown in SEQ ID NO.2. It was synthesized through the whole gene of Shanghai Jierui Bioengineering Co., Ltd., and the synthesized gene was cloned in the pUC57 plasmid (purchased from Shanghai Jierui Bioengineering Co., Ltd. ), the plasmid pUC57-lacMP was obtained.

Embodiment 2

[0030] Construction of Recombinant Plasmid pPICZαA-6AA-LacMP Containing Novel Laccase Gene and E.coli Top10 / pPICZαA-6AA-LacMP Carrying the Plasmid

[0031] Design PCR primers according to the nucleotide sequence of the novel laccase gene: forward primer PMPF (SEQ ID NO: 3): 5'-G GAATTC GCTATTGGACCAGTTGCT-3' (the EcoRI restriction site is underlined) and reverse primer PMPR (SEQ ID NO: 4): 5'-ATTT GCGGCCGC CAAATCAGA GGCTGGAGTAG-3' (the underline is the NotI restriction site). Design PCR primers according to the nucleotide sequence of the α signal peptide of pPICZαA: forward primer α-factor-6AAF (SEQ ID NO:5): 5'-TA T TCGAA ATGAGATTTCCTTCAATTTTTACT-3' (the underline is the BstBI restriction site) and reverse primer α-factor-6AAR (SEQ ID NO:6): 5'-G GAATTC AAACTCAGCCTCAGTCTCAGCTTCAGCCTCTCTTTTCTCG-3' (the underline is the EcoRI restriction site, and also contains the nucleotide sequence encoding 6 amino acids (ETEAEF)).

[0032] Using the plasmid pUC57-lacMP in Example 1 a...

Embodiment 3

[0040] Construction of a Novel Laccase-producing Strain Pichia pastoris X33 / pPICZαA-6AA-LacMP and Expression of Recombinant Laccase

[0041] The recombinant plasmid pPICZαA-6AA-LacMP was linearized by PmeI digestion and purified, then electrotransformed into Pichia pastoris X33 competent cells (purchased from Invitrogen Yingjie Life Technology Co., Ltd., USA), and placed on a YPD plate (1% yeast extract; 2 % tryptone; 2% glucose; 1.5% agar powder; containing 100 μg / L zeocin) for positive transformant screening. The transformant on the YPD plate was used as a template, and PMPF (SEQ ID NO: 3) and PMPR (SEQ ID NO: 4) were used as primers for PCR identification, and the correct transformant identified by PCR was inoculated on the MM plate (1.34% YNB; 4 ×10 -5 % Biotin; 0.5% Methanol; 1.5% Agar Powder; Contains 0.2mM ABTS and 0.1mM CuSO 4 ) for activity identification, the transformant with a dark green reaction circle around the colony is the recombinant Pichia pastoris X33 / pPI...

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Abstract

The invention discloses a moniliophthora perniciosa laccase and engineering bacteria, a recombinant laccase as well as application thereof. The amino acid sequence of the moniliophthora perniciosa laccase is shown as SEQ ID NO: 1. The DNA (Deoxyribonucleic Acid) sequence for encoding the laccase is shown as SEQ ID NO: 2. The DNA sequence is applied to construction of a pichia pastoris expression system. The recombinant pichia pastoris engineering bacteria is obtained by linearizing expression carrier pPICZalphaA-6AA-LacMP and then converting into the pichia pastoris. The recombinant laccase is obtained by fermenting the recombinant pichia pastoris engineering bacteria. The recombinant laccase has laccase activity and has higher tolerance to neutral or alkaline environments and halogens; application of the laccase to synthesis of ferulic acid copolymer through a biological enzyme method.

Description

technical field [0001] The invention belongs to the field of biotechnology and environmental biology, and particularly relates to a laccase gene of cocoa arbuscular fungus (Moniliophthora perniciosa) and the application of the gene engineering bacterium and the laccase coded by the gene in the synthesis of ferulic acid polymer. Background technique [0002] Laccase (benzonediol:oxygen oxidoreductases, EC1.10.3.2) is a copper-containing polyphenol oxidase, which belongs to the blue multicopper oxidase (MCOs) family and is widely distributed in plants, fungi, a few bacteria and insects. The active center of laccase generally contains 4 copper ions. The catalyzed oxidation reaction of laccase is to transfer electrons through the cooperation between copper ions, capture the electrons of the reaction substrate, oxidize the substrate into a free radical form, and capture the free radical from the substrate at the same time. electrons are passed to O 2 , will O 2 Revert to water....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P7/62C12R1/84
Inventor 林影刘慧平梁书利韩双艳郑穗平
Owner SOUTH CHINA UNIV OF TECH
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