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Bacillus subtilis ZNXH1 sourced bacterial laccase gene, bacterial laccase and application thereof

A technology of Bacillus subtilis and bacteria, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of industrial dye wastewater discharge, dye wastewater can not be effectively zero pollution, energy consumption, etc.

Active Publication Date: 2017-02-22
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The dye wastewater produced by industries such as textiles, papermaking, and plastics is often not effectively treated with zero pollution, resulting in the discharge of a large amount of industrial dye wastewater
Most of the synthetic dyes used in industry are aromatic compounds, and the traditional methods cannot effectively remove the dyes in the wastewater, and it is very energy-consuming, but in contrast, the application of laccase to treat industrial dye wastewater is economical, effective, and efficient. It is also environmentally friendly and is an ideal governance method at present and in the future

Method used

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  • Bacillus subtilis ZNXH1 sourced bacterial laccase gene, bacterial laccase and application thereof
  • Bacillus subtilis ZNXH1 sourced bacterial laccase gene, bacterial laccase and application thereof
  • Bacillus subtilis ZNXH1 sourced bacterial laccase gene, bacterial laccase and application thereof

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: the expression and the preparation of bacterial laccase CAHH1 gene cloning and bacterial laccase CAHH1 comprise the following steps:

[0027] (1) Cultivation of Bacillus subtilis ZNXH1 and extraction of chromosomal DNA

[0028] Inoculate the strain of Bacillus subtilis BS168 (available from Shanghai Beinuo Technology Co., Ltd., Beijing Tianchenze Biotechnology Co., Ltd., etc.) into 0.5 mL of LB liquid medium (the components of each 100 mL of medium are as follows: 0.5 g of yeast powder, 1.0 g peptone, 1.0g NaCl, the rest is water, pH = 7.6), 37°C, 180rpm, shaking culture for 2h, pour it into an empty petri dish, place it at 30cm of ultraviolet lamp (20W) and irradiate it for 20s with an interval of 20s , a total of 3 times of irradiation. Then take 100 μL of bacterial liquid and spread it on the LB agar culture plate (the components of each 100 ml medium are as follows: 0.5g yeast powder, 1.0g peptone, 1.0g NaCl, 0.5g agar powder, the rest is water, pH=7....

Embodiment 2

[0048] Embodiment 2: the characteristic of bacterial laccase CAHH1

[0049] (1) Optimum reaction pH and pH stability

[0050] pH value is an important factor affecting the catalytic activity of enzymes. Usually the enzyme shows the maximum catalytic activity at a certain pH value, and the farther it deviates from this pH value, the lower the catalytic activity of the enzyme. The maximum pH range detected by the catalytic activity of bacterial laccase CAHH1 of the present invention is 2.0-9.0 .

[0051] Using ABTS as a substrate, the activity of bacterial laccase CAHH1 in buffer solutions with different pH values ​​(2.0-9.0) was measured, and the relative enzyme activity under other pH conditions was calculated with the highest enzyme activity as 100%. The relative activity of the enzyme was plotted against the pH value ( Figure 5 A), the results show that the optimal pH value of bacterial laccase CAHH1 catalyzing ABTS is 4.6.

[0052] After incubating bacterial laccase CAHH...

Embodiment 3

[0057] Example 3: Co 2+ with Mn 2+ Effects on the Catalytic Activity of Bacterial Laccase CAHH1

[0058] With ABTS as substrate, cation Co 2+ and Mn 2+ The effect of (10mM) on the catalytic activity of bacterial laccase is shown in Figure 7, and the relative enzyme activity under other conditions was calculated with the enzyme activity of the control group as 100%. The results proved that: Co 2+ and Mn 2+ Both have the effect of enhancing the catalytic activity of bacterial laccase CAHH1.

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Abstract

The invention provides a bacillus subtilis ZNXH1 sourced bacterial laccase gene, bacterial laccase and application thereof, belongs to the technical field of biology, and particularly relates to a bacillus subtilis ZNXH1 sourced bacterial laccase gene cahh1, bacterial laccase CAHH1 expressed by the gene and application of the bacterial laccase CAHH1 in the fields of textile industry, environmental protection and the like. Compared with traditional laccase, the bacterial laccase CAHH1 has the advantages of remarkable application stability and broad spectrum effect, mainly including that (1) the bacterial laccase CAHH1 has better pH stability in a range of 3-9; (2) the bacterial laccase CAHH1 has better temperature stability in a range of 30-90 DEG C; and (3) the bacterial laccase CAHH1 has a strong decolorizing effect on indigo dye, azo dye and triphenylmethane dye. The bacterial laccase can be used for decolorizing industrial dye, has wide application prospects, can easily realize engineering preparation and has higher practical values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a bacterial laccase gene cahh1 derived from Bacillus subtilis ZNXH1, a bacterial laccase CAHH1 expressed by the gene, and applications of the bacterial laccase CAHH1 in the fields of textile industry and environmental protection. Background technique [0002] Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase, which can catalyze the oxidation of aromatic compounds with various structures by using copper ions in the active center, and at the same time reduce molecular oxygen to water. Because laccase has a wide range of substrates and high catalytic efficiency, it is widely used in paper industry (biological pulping and bleaching), textile industry (artificial dye decolorization), food industry (food flavor improvement), feed nutrition improvement, and new drugs. Development, soil bioremediation, biosensor manufacturing and new energy development and other fiel...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C02F3/34
CPCC02F3/342C12N9/0061C12Y110/03002
Inventor 张应玖金兰娜杨雪
Owner JILIN UNIV
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