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Laccase from Klebsiella pneumoniae, as well as recombinant strain and preparation method thereof

A Klebsiella and laccase technology, applied in the field of genetic engineering of enzymes, can solve the problem of less bacterial laccase, and achieve the effects of good thermal stability, large application potential, and good decolorization effect

Pending Publication Date: 2017-02-22
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on bacterial laccases, and more bacterial laccase genes with high activity and high stability need to be further explored and studied

Method used

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  • Laccase from Klebsiella pneumoniae, as well as recombinant strain and preparation method thereof
  • Laccase from Klebsiella pneumoniae, as well as recombinant strain and preparation method thereof
  • Laccase from Klebsiella pneumoniae, as well as recombinant strain and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Acquisition of Klebsiella pneumoniae Novel Laccase Mature Peptide Gene

[0048] 1. The new laccase mature peptide gene is derived from Klebsiella pneumoniae screened in our laboratory, and its genomic DNA is extracted. The extraction steps of Klebsiella pneumoniae genomic DNA are as follows:

[0049] (1) Inoculate and streak on LB solid plates from glycerol tubes, and culture at 37°C for 12 hours;

[0050] (2) Pick a single colony from the culture plate and inoculate it in 5mL liquid LB medium, and culture it at 220r / min, 37°C for 12h;

[0051] (3) Dispense the bacterial liquid into sterilized 1.5mL microcentrifuge tubes, centrifuge at 12000r / min for 1min to collect the bacterial cells, and discard the supernatant;

[0052] (4) Resuspend the precipitate in 200 μL of pre-cooled solution I and repeatedly pipette and mix with a pipette tip, then add 50 μL of 50 mg / mL lysozyme, and bathe in water at 37°C for 1 hour;

[0053] (5) Add 20 μL of 10% SDS and 10 μL o...

Embodiment 2

[0071] Example 2: Construction of a novel laccase recombinant bacterium with high stability of Bacillus subtilis

[0072] 1. Construction of expression vector pBSA43

[0073] pBSA43 is obtained by using the E. coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloning into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium . it comes with amp r Gene that can use ampicillin resistance as a selectable marker in E. coli; also has Km r Gene, kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis.

[0074] 2. Construction of a novel laccase expression vector pBSA43-Lac

[0075]The novel laccase gene (Lac) amplified by PCR and recovered after double digestion with BamH I and Hind III was ligated with the same double digestion Bacillus subtilis expression vector pBSA43, and the ligated product was transformed int...

Embodiment 3

[0078] Example 3: Construction of recombinant strains for the free expression of novel laccase from Pichia pastoris

[0079] 1. Construction of a novel laccase expression vector pPIC9K-Lac

[0080] The novel laccase gene (Lac) amplified by PCR and recovered after EcoR I and Not I double-digestion was ligated with the same double-digestion Pichia expression vector pPIC9K with ligase; the ligated product was transformed into Escherichia coli JM109 In the competent cells, select positive transformants through Amp resistance screening; extract positive transformant plasmids, extract plasmids after shaking tube culture at 37°C, and conduct preliminary verification of single and double enzyme digestion, and verify the correct enzyme digestion The recombinant plasmid was named pPIC9K-Lac; the positive clones verified by enzyme digestion were sent to Beijing Huada Gene Technology Co., Ltd. for sequencing to further ensure the correctness of the target gene, and finally confirm the con...

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Abstract

The invention belongs to the technical field of gene engineering of enzymes and relates to a novel laccase from Klebsiella pneumoniae and a preparation method thereof. The invention adopts the technical scheme comprising the following steps of: utilizing a molecular biology method and a gene engineering technology to obtain a plant of Klebsiella pneumoniae capable of generating the laccase by strain screening, amplifying genes of the novel laccase by a PCR technology, and then carrying out expression on the genes of the novel laccase in a bacillus subtillis expression system and a pichia pastoris expression system respectively to obtain a high-stability laccase recombinant strain of bacillus subtillis and a high-stability laccase free expression recombinant strain of pichia pastoris, thereby realizing preparation of the novel laccase. Simultaneously, the novel bacterial laccase has a better decoloring effect for azo and anthraquinone dyes.

Description

[0001] Technical field: [0002] The present invention relates to a novel laccase derived from Klebsiella pneumoniae and its gene, engineering bacteria and preparation method, in particular to obtaining a recombinant expression strain expressing the novel laccase by means of genetic engineering technology and molecular biology, and The application of the bacterial laccase protein in the dye decolorization industry belongs to the technical field of enzyme genetic engineering. [0003] Background technique: [0004] Laccase (Laccase, E.C.1.10.3.2), also known as urushiol oxidase and multi-copper oxidase, is a copper-containing polyphenol oxidase, which is compatible with ascorbate oxidase in plants and ceruloplasmin in mammals. , Cytochrome C oxidase, and bilirubin oxidase are homologous and belong to the blue multi-copper oxidase family. It can catalyze the oxidation of various phenolic and non-phenolic compounds to generate corresponding quinones, and at the same time reduce m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/75C12N15/81C12N1/21C12N1/19C02F3/34C12R1/125C12R1/84
CPCC12N9/0061C02F3/342C12N15/75C12N15/815C12N2800/101C12N2800/102C12Y110/03002
Inventor 刘逸寒黄琳路福平郭伟
Owner TIANJIN UNIV OF SCI & TECH
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