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Bacterial laccase and gene, preparation method and application thereof

A laccase and bacteria technology, applied in the field of genetic engineering of enzymes, can solve the problems of high use cost, high medium requirements, long growth cycle of filamentous fungi, etc., and achieve a large application potential, stable vitality, and good decolorization effect Effect

Active Publication Date: 2019-09-10
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, industrialized commercial laccases are mainly derived from fungi, but generally fungal laccases maintain high activity under acidic conditions (pH 4-6) and 30-55°C, while industrial fields such as papermaking and textiles are often accompanied by high temperature. , strong alkali and other reaction conditions, the application of fungal laccase is limited and the cost of use is high
Moreover, filamentous fungi have a long growth cycle, have high requirements on the medium, and are susceptible to high mechanical damage in the fermenter. These problems have seriously affected the industrial application of fungal laccase.

Method used

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  • Bacterial laccase and gene, preparation method and application thereof
  • Bacterial laccase and gene, preparation method and application thereof
  • Bacterial laccase and gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Acquisition of a novel laccase mature peptide gene from Bacillus amyloliquefaciens

[0058] 1. The new laccase mature peptide gene is derived from the Bacillus amyloliquefaciens screened in our laboratory, and its genomic DNA is extracted using a kit (OMEGA: Bacterial DNA Kit). The extraction steps of the genomic DNA of Bacillus amyloliquefaciens are as follows:

[0059] (1) Inoculate and streak on LB solid plates from glycerol tubes, and culture at 37°C for 12 hours;

[0060] (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;

[0061] (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;

[0062] (4) Resuspend the pellet in 100 μL TE Buffer / molecular water and repeatedly blow and mix with a pipette tip, then add 50 μL of 50 mg / mL lysozyme, and place it in a 37...

Embodiment 2

[0082] Example 2: Construction of a novel laccase recombinant bacterium with high stability of Bacillus subtilis

[0083] 1. Construction of expression vector pBSA43

[0084] 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.

[0085] 2. Construction of a novel laccase expression vector pBSA43-Lac

[0086] The novel laccase gene (Lac) amplified by PCR and recovered after double digestion with BamHI and SalI was ligated with the same double digestion of Bacillus subtilis expression vector pBSA43 with ligase, and the ligated product was tran...

Embodiment 3

[0089] Example 3: Construction of a novel laccase recombinant bacterium with high stability of Bacillus amyloliquefaciens

[0090] 1. Construction of expression vector pBSA43

[0091] pBSA43 is based on the Escherichia coli-Bacillus amyloliquefaciens shuttle cloning vector pBE2 as the backbone, cloned into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium. get. 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.

[0092] 2. Construction of a novel laccase expression vector pBSA43-Lac

[0093] The novel laccase gene (Lac) amplified by PCR and recovered after double digestion with BamHI and SalI was ligated with the Bacillus amyloliquefaciens expression vector pBSA43 with the same double digestion, and the ...

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Abstract

The invention belongs to the technical field of genetic engineering of enzymes, and relates to a novel laccase from bacillus amyloliquefaciens and a gene, preparation method application of the novel laccase. According to the technical scheme, by using a molecular biology means and a genetic engineering technology, the bacillus amyloliquefaciens capable of generating the laccase is obtained throughstrain selection, the gene of the novel laccase is amplified through a PCR technique, and then the gene of the novel laccase is expressed in a bacillus subtillis expression system, a bacillus amyloliquefaciens expression system and a bacillus licheniformis expression system to obtain a bacillus subtillis high-stability laccase recombined strain, a bacillus amyloliquefaciens high-stability laccaserecombined strain and a bacillus licheniformis high-stability laccase free expression recombined strain respectively, and preparation of the novel laccase is achieved. The novel bacterial laccase plays a good role in the aspects of protein polymerization, dye decolorization and paper making.

Description

[0001] Technical field: [0002] The invention relates to a novel laccase derived from Bacillus amyloliquefaciens and its gene, preparation method and application, in particular to a recombinant expression strain expressing the novel laccase obtained through genetic engineering technology and molecular biology means, and the bacterial lacquer The application of enzyme protein in protein cross-linking, dye decolorization, and biological pulping belongs to the technical field of enzyme genetic engineering. [0003] Background technique: [0004] Laccases (diphenol:oxygen oxidoreductases, EC 1.10.3.2) are a family of multi-copper oxidases capable of oxidizing a variety of inorganic and aromatic compounds, especially phenols, while reducing molecular oxygen to water. Laccase not only has a wide range of catalytic substrates, but also has the ability to oxidize highly refractory and environmentally harmful compounds, and also has special oxidation catalytic properties. Compared wit...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/75C12N1/21C12R1/125C12R1/07C12R1/10
CPCC12N9/0061C12N15/75C12Y110/03002
Inventor 刘逸寒王洪彬路福平李艳珍李玉
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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