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Laccase CotA and application thereof

A laccase and DNA sequence technology, applied in the field of CotA laccase and its application, and the preparation of bacterial laccase, can solve the problems of slow expression and growth cycle of fungi, easy to produce inclusion bodies and the like, achieve good tolerance and promote enzyme activity. Effect

Inactive Publication Date: 2016-10-26
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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  • Laccase CotA and application thereof
  • Laccase CotA and application thereof
  • Laccase CotA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cloning and expression purification of embodiment 1 CotA laccase gene

[0033] 1. Cloning of CotA laccase gene

[0034] The pure colony of Bacillus subtilis cjp3 (CICC No. 24168) was picked into 5mL LB liquid medium, cultivated overnight at 37°C and 200rpm. Genomic DNA of the above strains was extracted by CTAB / NaCl method, and detected by 1% agarose gel electrophoresis.

[0035]Using genomic DNA as a template, the upstream primer CotA-F: 5′-ATAAGAATGCGGCCGCATGACACTTGAAAAATTTG-3′ (containing the Not I restriction site) and the downstream primer CotA-R: 5′-CCGCTCGAGTTATTTATGGGGATCAGTT-3′ (containing the Xho I restriction site Dots) CotA laccase gene of strain cjp3 was amplified.

[0036] PCR system: ddH 2 O 22 μL, 10 × Fast Pfu Buffer 10 μL, 10 mmol / L dNTP mixture 4 μL, PCR stimulate 10 μL, 100 mmol / L upstream and downstream primers 1 μL, DNA template 1 μL, 2.5 U / L Fast Pfu enzyme 1 μL.

[0037] The amplification program was: 98°C for 3min pre-denaturation, 95°C for ...

Embodiment 2

[0044] The properties of embodiment 2 recombinant laccase

[0045] 1. Effect of pH on CotA laccase activity

[0046] 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.

[0047] 2. The effect of temperature on the ac...

Embodiment 3

[0061] Embodiment 3 recombinant laccase is to the decolorization of synthetic dyestuff

[0062] 1. The reaction system of the decolorization experiment and the calculation of the decolorization rate

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Abstract

The invention discloses laccase CotA and application thereof. DNA (deoxyribonucleic acid) sequences of the laccase CotA are shown as SEQ ID NO.2, and amino acid sequences of the laccase CotA are shown as SEQ ID NO.1. The laccase CotA and the application have the advantages that the prepared laccase CotA is mature in operation, easy and convenient to operate, high in enzyme activity and wide in pH (potential of hydrogen) and temperature catalysis range, is good in stability in high-salinity and high-concentration organic solvents under alkaline and high-temperature conditions and is high in applicability as compared with fungal laccase; synthetic dye with different chemical structures can be effectively decolorized by the laccase CotA under the condition of participation of acetosyringone which is a mediator substance, excellent decolorization effects still can be kept under alkaline conditions, and accordingly the laccase CotA has an excellent application prospect in the aspect of treatment on industrial dye wastewater.

Description

technical field [0001] The invention belongs to the aspect of applied microorganisms, and relates to the preparation of a bacterial laccase, in particular to CotA laccase and its application. 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 is a small molecule tha...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C02F3/34
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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