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A Laccase-Producing Mutant Thromba Trametes and Its Application

A technology of Trametes rubrum and laccase, applied in the direction of enzymes, fungi, oxidoreductase, etc., can solve the problems of high price and limited application of laccase

Active Publication Date: 2022-05-24
河南新仰韶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are not many actual laccase products on the market, or the price is relatively high. The main reason is that the extraction of laccase from plants is restricted by the growth rate and growth of tree species, and when it is extracted from fungi, it is limited by fungal fermentation. The low yield is restricted, thus limiting the application of laccase in various fields

Method used

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  • A Laccase-Producing Mutant Thromba Trametes and Its Application

Examples

Experimental program
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specific Embodiment

[0024] The application will be further explained below in conjunction with the examples. Before introducing specific embodiments, some experimental backgrounds involved in the following embodiments are briefly described as follows.

[0025] Experimental reagents:

[0026] 10mmol / L 2,6-dimethoxyphenol (DMP), 0.1mol / L citric acid phosphate buffer (pH3.5), conventionally prepared; however, it should be noted that 2,6-dimethylformaldehyde is prepared After the solution of oxyphenol (DMP), it needs to be stored in the refrigerator in a brown bottle for one week before use.

[0027] Determination of laccase enzyme activity (DMP method)

[0028] The definition of enzymatic activity is: under certain temperature and pH conditions, the amount of enzyme required to produce 1 μmol of product by oxidation within 1 min is one enzymatic activity unit;

[0029] Since laccase can oxidize the substrate 2,6-dimethoxyphenol (DMP) to 2,6-dimethoxyphenolquinone, the substance has a maximum abso...

Embodiment 1

[0037] In this example, the mutagenesis, selection and acquisition process of the Trametes rubrum YSQ8 strain provided in this application is briefly introduced as follows.

[0038] The T. rubrum YSQ8 strain provided in this application is based on the existing two strains of T. rubrum with different phenotypes (YSQ6-105, YSQ7), and is obtained by plastid fusion and directional breeding. The specific process is briefly described as follows.

[0039] (1) Preparation of protoplasts

[0040] Take the two preserved starting strains (YSQ6-105, YSQ7), suspend them with 0.6M sorbitol osmotic pressure stabilizer (0.1M citric acid buffer+0.6M sorbitol+0.01M EDTA), and put them on ice for later use;

[0041] Then add lysozyme 1.2%, helicase 0.8%, cellulase 0.8%, and enzymolysis at 30°C for 6 hours to prepare protoplasts for later use;

[0042] In the preparation process, the common medium for protoplast culture is: potato 200g, glucose 20g, agar 20g, water 1000mL; the regeneration medi...

Embodiment 2

[0063] On the basis of the strain obtained by screening in Example 1, the inventor further optimized the fermentation process to which it was adapted to further improve the fermentation enzyme production capacity of the strain. The specific process is introduced as follows.

[0064] (1) Strain activation and preparation of seed solution

[0065] The preserved Trametes rubrum YSQ8 mycelium was inoculated in a PDA culture bottle, fermented and cultured at 30° C., 220rpm for 5d, transferred to a sterile bottle, and the seed bottle of the seed tank was obtained;

[0066] Then carry out the preparation of the first-class seed liquid, specifically: inoculate the seed bottle into the PDA medium of the first-class seed tank, and ferment for 56h at 30°C and 180rpm;

[0067] Then, the secondary seed liquid was prepared, specifically: the primary seed liquid was inoculated into the PDA medium of the secondary seed tank under aseptic conditions, and fermented at 30° C. and 160 rpm for 50...

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Abstract

The invention belongs to the technical field of biological fermentation engineering, and specifically relates to a laccase-producing mutant bacterium Thrombus rubrum and its application patent application. Thrombocytosis YSQ8 strain was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on December 5, 2019. The preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation number for CGMCC 19142. In this application, on the basis of the existing two strains of Thrombus erythrorrhagia (YSQ6-105, YSQ7) with different phenotypes, through ultraviolet mutagenesis and protoplast fusion, the inventor further obtained the A strain of Thrombus Rhodopsin YSQ8 with high fermentative enzyme activity. Preliminary experimental results show that the fermentation enzyme activity of YSQ8 strain can be increased by about 27 times compared with the original starting strain, showing a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological fermentation engineering, and particularly relates to a laccase-producing mutant bacterium Rheumatoid fungus and a patent application for its application. Background technique [0002] Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase that can catalyze the oxidation of polyphenols, polyaminobenzenes and other substances to generate corresponding quinones and water. Laccase was first discovered by Japanese scholar Yoshida from the Japanese Lac lacquer tree ( Rhus vernicifera ), and was named a metal-containing oxidase by Bertrand in 1894. In 1986, Bertrand and Laborde et al. found that this enzyme also exists in higher fungi. [0003] Laccase has a wide range of sources. According to the source, it is divided into two categories: laccase from laccase and fungal laccase. Fungal laccase has strong catalytic performance and can degrade lignin and phenolic substances. Therefore, it...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/02C12R1/645
CPCC12N1/14C12N9/0061C12Y110/03002C12R2001/645C12N1/145
Inventor 孙利鹏管宁焦国宝陈晓宇梁颖杰卢佳琪邹瑞瑞
Owner 河南新仰韶生物科技有限公司
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