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A kind of tyrosinase mutant and its application

A tyrosinase and mutant technology, which is applied in the field of tyrosinase mutants and the synthesis of theaflavins using them, and achieves the effects of high catalytic efficiency, high catalytic efficiency and conversion yield, and high specific activity

Active Publication Date: 2022-05-24
HUNAN FLAG BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently commercialized tyrosinase, whose enzyme source comes from fungal microorganisms such as mushrooms, has also been reported on the application of tyrosinase to prepare theaflavins.

Method used

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  • A kind of tyrosinase mutant and its application
  • A kind of tyrosinase mutant and its application
  • A kind of tyrosinase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of Bacillus megaterium-derived tyrosinase Bmtyrc prokaryotic expression strain

[0037] Download the amino acid sequence of tyrosinase derived from Bacillus megaterium in GenBank (SEQ ID NO.1 in this paper, corresponding to GenBank accession number: ACC86108.1), and submit the amino acid sequence to Beijing Qingke Biotechnology Co., Ltd. for the synthesis of the whole gene sequence (using large intestine Bacillus preferred codons). The C-terminal of the synthetic gene carries a His tag and is constructed into the prokaryotic expression vector pET30a(+). Pass the constructed plasmid pET30a(+)-Bmtyrc through CaCl 2 Transform into Escherichia coli expression strain BL21(DE3) by heat shock transformation, spread on LB solid medium plate containing 50 μg / ml Kanamycin, cultivate overnight at 37°C, and the colonies grown on the plate are prokaryotic expression of tyrosinase Recombinant strain E. coli BL21(DE3) / pET30a(+)-Bmtyrc.

[0038] Use a sterili...

Embodiment 2

[0039] Example 2: Purification and Immobilization of Tyrosinase (Bmtyrc)

[0040] Using the His tag carried in the Bmtyrc recombinant protein, using activated IDA resin (purchased from Anoron (Beijing) Biotechnology Co., Ltd., specific model: His.Bind Resin, Ni-charged), the specific methods and steps used As follows: 4°C, 10000r / min, centrifuge the fermentation broth for 10min, discard the supernatant, collect the cells, wash the cells twice with phosphate buffer (pH8.0, 0.1mol / L), and concentrate the cells after centrifugation 5-fold resuspended in 20 mL of phosphate buffer (pH 8.0, 0.1 mol / L). The bacterial solution after the above treatment was placed in ice water for sonication until clarification, and the sonication conditions were: working for 2s, interval of 5s, and ultrasonic power of 500W. The fragmented lysate was centrifuged in a low-temperature high-speed centrifuge (12000 rpm, 4° C., 20 min), and the supernatant was collected to obtain crude protein. The crude ...

Embodiment 3

[0044] Example 3: Construction of Bmtyrc prokaryotic expression strain E. coli BL21(DE3) / pET30a(+)-Bmtyrc error-prone mutation library

[0045] Using pET30a(+)-Bmtyrc recombinant plasmid as PCR template, conventional T7F / R as universal primer (primer sequence: T7F: 5'-TAATACGACTCACTATAGGG-3', T7R: GCTAGTTATTGCTCAGCGG see SEQ ID NO. 12 and 13) for Bmtyrc gene Perform error-prone PCR amplification and adjust Mg in the PCR amplification reaction system 2+ , Mn 2+ , dCTP and dTTP oligonucleotide concentrations, so that the base mismatch rate of the mutant library is only 2 / 1000, that is, to ensure that only 1 to 2 amino acids are mutated in a mutant.

[0046] Error-prone PCR reaction system:

[0047]

[0048] Error-prone PCR reaction conditions: pre-denaturation at 95°C for 5 min; then denaturation at 94°C for 30 s, annealing at 56°C for 1 min, extension at 72°C for 1.5 min, a total of 25 cycles; and finally extension at 72°C for 10 min.

[0049] 2 μL of the above error-pron...

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Abstract

The invention belongs to the technical field of enzyme engineering and relates to a tyrosinase mutant and its application. The tyrosinase mutant has multiple amino acid positions mutated in the wild-type tyrosinase in the amino acid sequence shown in SEQ ID NO.1. Utilizing the tyrosinase mutant and the method for preparing theaflavin of the present invention can make the mutant have higher specific activity than wild-type tyrosinase, and have higher yield when catalyzing the preparation of theaflavin.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and relates to a tyrosinase mutant and the application of synthesizing theaflavin using the mutant. Background technique [0002] In China, tea, as the national drink, has a long cultural and historical origin. According to the production and properties of tea, it can be divided into six major types of tea: black tea, green tea, green tea, yellow tea, dark tea, and white tea. As the second largest tea category in my country, black tea is produced in a wide range of regions, including Fujian, Guangdong, Yunnan, Taiwan and other more than ten provinces, Yunnan and Fujian are the main planting areas. The most obvious feature that distinguishes black tea from other teas is that it has the characteristics of black tea, red soup, red leaves, sweet and mellow, and is deeply loved by consumers. In recent years, studies have shown that black tea has many physiological functions that are beneficial to he...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N11/089C12P17/16
CPCC12N9/0071C12N11/089C12P17/162C12Y114/18001
Inventor 周晶辉刘仲华张盛刘昌伟赵士敏刘亚赵强许岗
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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