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Bacterial laccase gene from bacillus subtilis ZXN4, 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 dye wastewater that cannot be effectively zero-polluted, treated, and consume energy

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

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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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  • Bacterial laccase gene from bacillus subtilis ZXN4, bacterial laccase and application thereof
  • Bacterial laccase gene from bacillus subtilis ZXN4, bacterial laccase and application thereof
  • Bacterial laccase gene from bacillus subtilis ZXN4, 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 CAH4 gene cloning and bacterial laccase CAH4 comprise the following steps:

[0027] (1) Cultivation of Bacillus subtilis ZXN4 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 0.5ml of LB liquid medium (the components of each 100ml medium are as follows: 0.5g 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 5 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=7.6) After...

Embodiment 2

[0048] Example 2: Characterization of Bacterial Laccase CAH4

[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 CAH4 of the present invention is 2.0-9.0 .

[0051]Using ABTS as a substrate, the activity of bacterial laccase CAH4 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 CAH4 catalyzing ABTS is 4.6.

[0052] After incubating the bacterial laccase CAH4 in ...

Embodiment 3

[0057] Example 3: Co 2+ with Mn 2+ Effects on Catalytic Activity of Bacterial Laccase CAH4

[0058] With ABTS as substrate, cation Co 2+ and Mn 2+ (10mM) on the catalytic activity of bacterial laccase Figure 7 As indicated, 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 CAH4.

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Abstract

The invention discloses a bacterial laccase gene from bacillus subtilis ZXN4, bacterial laccase and an application thereof, belongs to the technical field of biology, and particularly relates to a bacterial laccase gene cah4 from the bacillus subtilis ZXN4, bacterial laccase CAH4 expressed by the bacterial laccase gene cah4 and the application of the bacterial laccase CAH4 to the fields of textile industry, environment protection and the like. Compared with traditional laccase, the bacterial laccase CAH4 has the advantages of being obvious in application stability and effect wide spectrum, and mainly has the advantages that the bacterial laccase CAH4 has the good acidic and alkaline stability within the range of pH equal to 3 to 7, the good temperature stability within the temperature range of 30 DEG C to 90 DEG C and the high decoloration effect on indigo blue dye, azo dye and triphenylmethane dye, can be used for decoloration of industry dye, and has broad application prospects, engineering preparing is easy to achieve, and the bacterial laccase CAH4 has the high practical value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a bacterial laccase gene cah4 derived from Bacillus subtilis ZXN4, a bacterial laccase CAH4 expressed by the gene, and applications of the bacterial laccase CAH4 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 fields ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C02F3/34C02F101/30
CPCC02F3/342C02F2101/308C12N9/0061C12Y110/03002
Inventor 张应玖杨雪金兰娜
Owner JILIN UNIV
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