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Bacillus subtilis cjp3 for producing CotA laccase as well as CotA laccase and application thereof

A technology of Bacillus subtilis and laccase, which is applied in the field of applied microorganisms, can solve the problems of slow expression growth cycle of fungi and easy generation of inclusion bodies, etc., and achieves the effects of strong applicability, good stability, and mature and convenient operation.

Pending Publication Date: 2017-05-31
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial laccase is usually expressed in Escherichia coli and yeast, but the growth cycle of fungal expression is slow, the operation is not as convenient as Escherichia coli, and the expression level is not as high as the Escherichia coli system
At present, the most widely used and most mature expression system is the large intestine expression system, but the E. coli expression system is prone to produce inclusion bodies

Method used

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  • Bacillus subtilis cjp3 for producing CotA laccase as well as CotA laccase and application thereof
  • Bacillus subtilis cjp3 for producing CotA laccase as well as CotA laccase and application thereof
  • Bacillus subtilis cjp3 for producing CotA laccase as well as CotA laccase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning and expression purification of CotA laccase gene

[0037] 1. Cloning of the CotA laccase gene

[0038] The pure colony of Bacillus subtilis cjp3 (CCTCC NO: M 2016408) was picked into 5 mL LB liquid medium, cultivated overnight at 37°C and 200 rpm. Genomic DNA of the above strains was extracted by CTAB / NaCl method, and detected by 1% agarose gel electrophoresis.

[0039] Use genomic DNA as a template and upstream primer CotA-F:

[0040] 5′-ATAAGAATGCGGCCGCATGACACTTGAAAAATTTG-3′ (containing Not I restriction site) and downstream primer CotA-R: 5′-CCGCTCGAGTTATTTATGGGGATCAGTT-3′ (containing Xho I restriction site) amplified the CotA laccase gene of strain cjp3.

[0041] PCR system: ddH 2 O 22μL, 10×Fast Pfu Buffer 10μL, 10mmol / L dNTP mixture 4μL, PCR stimulate 10μL, 100mmol / L upstream and downstream primers 1μL, DNA template 1μL, 2.5U / L Fast Pfu enzyme 1μL.

[0042] The amplification program was: 98°C for 3min pre-denaturation, 95°C for 30s, 55°C for ...

Embodiment 2

[0049] The properties of embodiment 2 recombinant laccase

[0050] 1. The effect of pH on CotA laccase activity

[0051] The effect of pH on laccase activity was measured by 0.1M citric acid-phosphate buffer (pH 3.0-7.0), 0.1M Tris-HCl buffer (pH7.0-9.0). The optimum pH of laccase enzyme activity was measured with ABTS in 0.1M citric acid-phosphate buffer (pH 3.0-7.0). The effect of pH on the stability of bacterial laccase was determined by measuring the remaining enzyme activity after incubation at 30°C for several hours at pH 3.0, 7.0, 9.0. The result is as figure 1 As shown, it shows that the CotA laccase provided by the present invention has a wide range of catalysis, and can catalyze substrate reactions in the range of pH 3-9. When ABTS was used as the substrate, the optimum pH measured was 5, figure 2 It shows that CotA laccase has good stability in the environment of pH9.0, and can still maintain high activity after 10h.

[0052] 2. The effect of temperature on th...

Embodiment 3

[0064] Embodiment 3 recombinant laccase is to the decolorization of synthetic dyestuff

[0065] 1. The reaction system of the decolorization experiment and the calculation of the decolorization rate

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Abstract

The invention discloses a bacillus subtilis cjp3 for producing CotA laccase as well as CotA laccase and application thereof. The CotA laccase producing bacterium is named as Bacillus subtilis, collected in China Center for Type Culture Collection with number CCTCC No: M 2016408 on July 21, 2016 in Wuhan University of China. The DNA sequence of the CotA laccase is shown as SEQ ID NO. 2, and the amino acid sequence is shown as SEQ ID NO. 1. The operation on the CotA laccase is mature and convenient, the obtained laccase has high activity, and the prepared CotA laccase has a wide pH and temperature catalysis range, high stability under alkali and high-temperature conditions in the presence of high salt and high-concentration organic solvents, and stronger practicability than fungal laccase. Under the participation of an acetosyringone mediator, the CotA laccase can be used for effectively decolorizing synthetic dyes of different chemical structures, still keeps a good decolorizing effect under the alkali condition, and has a good application prospect on treatment of industrial dye wastewater.

Description

technical field [0001] The invention belongs to the technical field of applied microorganisms, and in particular relates to a CotA laccase-producing bacillus subtilis cjp3, its CotA laccase and applications thereof. Background technique [0002] Laccase (diphenol oxidase, EC 1.10.3.2), a member of the polycopper oxidation family, catalyzes the oxidation of a variety of phenolic and nonphenolic substrates, such as phenol, chlorophenol, diphenylmethane, benzo Ratio, m-phenylenediamine, etc. Due to the wide range of substrates catalyzed by laccase oxidation, it is used in many industries, including food processing plants, paper mills, textile mills, paint plants, and organic chemical plants. [0003] Laccases are typical multi-copper oxidases involved in (1) cross-linking of monomers, (2) degradation of polymers, and (3) ring-opening of aromatic compounds. When catalyzing the oxidation of non-phenolic compounds, laccase requires the presence of a mediator in the system, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/02C12N15/53C02F3/34C12R1/125C02F103/34
CPCC02F3/342C02F2103/34C12N9/0061C12Y110/03002C12N1/205C12R2001/125
Inventor 乔维川褚靖萍夏昊谢震宇
Owner NANJING FORESTRY UNIV
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