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Thermus thermophilus laccase (benzenediol: oxygen oxidoreductases), engineering bacteria, recombinant laccase and use of recombinant laccase

A Thermus thermophilus, engineering bacteria technology, applied in application, genetic engineering, recombinant DNA technology and other directions, can solve problems such as high cost and poor effect, and achieve good heat and alkali resistance, high tolerance. Effect

Active Publication Date: 2014-08-27
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most industrial dyes are difficult to be degraded by microorganisms, and the use of physical or chemical methods is often costly and ineffective

Method used

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  • Thermus thermophilus laccase (benzenediol: oxygen oxidoreductases), engineering bacteria, recombinant laccase and use of recombinant laccase
  • Thermus thermophilus laccase (benzenediol: oxygen oxidoreductases), engineering bacteria, recombinant laccase and use of recombinant laccase
  • Thermus thermophilus laccase (benzenediol: oxygen oxidoreductases), engineering bacteria, recombinant laccase and use of recombinant laccase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Synthesis of Novel Laccase Gene

[0030] Based on the GenBand: YP_005641270.1 amino acid sequence published on the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ), the sequence is based on the codon preference of Pichia pastoris Optimization was carried out to obtain a novel laccase gene whose sequence is shown in SEQ ID NO.1, which was synthesized by a biotechnology company (Nanjing Kingsray Biotechnology Co., Ltd.), and the synthesized gene was cloned in the pUC57 plasmid (purchased from Kings Rui Biological Technology Co., Ltd.) to obtain the plasmid pUC57-lacTT.

Embodiment 2

[0032] Construction of Recombinant Plasmid pHKFA1-lacTT Containing Novel Laccase Gene and E.coli Top10 / pHKFA1-lacTT Carrying the Plasmid

[0033] Design PCR primers according to the nucleotide sequence of the novel laccase gene: forward primer TTF: 5'-G GAATTC AATACCGATAGAAGAACCCT-3' (underlined EcoRI restriction site) and reverse primer TTR: 5'-ATTT GCGGCCGC TTAGTGGTGGTGGTGGTGGTGGTGGTG TCCGACTTCCAAAACTCC-3' (the underline is the NotI restriction site, which also contains 8×His tag and stop codon).

[0034] The plasmid pUC57-lacTT in Example 1 was used as a template, and TTF and TTR were used as primers for PCR amplification. The gel-recovered and purified PCR product was double-digested with restriction endonucleases EcoRI and NotI, and ligated with the plasmid pHKFA1 that had also been double-digested with EcoRI and NotI with T4 ligase overnight at 16°C, and the ligated product was chemically transformed into E.coli Top10 (purchased from Invitrogen Life Technology Co., L...

Embodiment 3

[0036] Construction of a Novel Laccase-producing Strain Pichia pastoris GS115 / pHKFA1-lacTT and Expression of Recombinant Laccase

[0037]The recombinant plasmid pHKFA1-lacTT was linearized and purified by Kpn2I single enzyme digestion, and then electrotransformed into Pichia pastoris GS115 (purchased from Invitrogen Life Technologies Co., Ltd., USA) competent cells, and positive transformants were screened on MD plates. The transformant screened on the MD plate was used as a template, and TTF and TTR were used as primers for PCR identification. The correct transformant identified by PCR was initially identified as a new laccase-producing strain Pichia pastoris GS115 / pHKFA1-lacTT.

[0038] Pick 3 transformants identified correctly by PCR and inoculate them into BMGY medium, and culture them at 30°C and 250rpm until OD 600 Close to 6.0, collect the cells by centrifugation at 6000rpm, 4°C for 5min, and then resuspend the collected cells in BMMY medium (containing 0.1mM CuSO 4 ) ...

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Abstract

The invention discloses a thermus thermophilus laccase (benzenediol: oxygen oxidoreductases), engineering bacteria, a recombinant laccase and a use of the recombinant laccase. The thermus thermophilus laccase has an amino acid sequence shown in the formula of Seq No.2. The DNA sequence for coding the above laccase is shown in the formula of Seq No.1. The DNA can be used in construction of a Pichia pastoris expression system. Through expression vector pHKFA1-lacTT linearization and then Pichia pastoris conversion by the expression vector, recombinant Pichia pastoris engineering bacteria are obtained. The recombinant laccase is obtained by fermentation of the recombinant Pichia pastoris engineering bacteria. The recombinant laccase has laccase activity and high temperature resistance, can be used in dye decoloring and especially in azo dye and anthraquinone dye decoloring, and has a decolourization ratio of 100%.

Description

technical field [0001] The invention relates to the fields of biotechnology and environmental biology, in particular to a thermophilic bacteria laccase gene and the application of the genetically engineered bacterium and the laccase coded by the gene in biological enzymatic treatment of textile wastewater. Background technique [0002] Laccase (benzenediol:oxygen oxidoreductases, EC1.10.3.2) is a copper-containing polyphenol oxidase, which belongs to the family of blue multicopper oxidases (MCOs), 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. Laccase acts on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C02F3/00C12R1/84
CPCC12N9/0061C12N15/815C12Y110/03002C02F3/34C12N15/81C12N1/165C12R2001/84
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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