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Tyrosinase mutant and application thereof

A kind of tyrosinase and mutant technology, applied in the field of tyrosinase mutants and the synthesis of theaflavins using the same

Active Publication Date: 2021-09-10
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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  • Tyrosinase mutant and application thereof
  • Tyrosinase mutant and application thereof
  • Tyrosinase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0037] Download the amino acid sequence of tyrosinase derived from Bacillus megaterium in GenBank (SEQ ID NO.1 in this article, corresponding to GenBank accession number: ACC86108.1), and submit the amino acid sequence to Beijing Qingke Biotechnology Co., Ltd. for complete gene sequence synthesis (using large intestine Bacillus preferred codon). The C-terminal of the synthetic gene has a His tag, and it is constructed into the prokaryotic expression vector pET30a(+). The prokaryotic expression vector restriction site: Nde I at the 5' end, Xho I at the 3' end. Pass the constructed plasmid pET30a(+)-Bmtyrc through CaCl 2 Transformed into Escherichia coli expression strain BL21(DE3) by heat shock transformation method, spread on LB solid medium plate containing 50 μg / ml Kanamycin, and cultivate overnight at 37°C, the colony grown on the plate is prokaryotic exp...

Embodiment 2

[0039] Embodiment 2: Purification and immobilization of tyrosinase (Bmtyrc)

[0040] Using the His tag carried in the Bmtyrc recombinant protein, using activated IDA resin (purchased from Anolun (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 bacteria, wash the bacteria twice with phosphate buffer (pH8.0, 0.1mol / L), concentrate the bacteria after centrifugation 5 times resuspended in 20mL phosphate buffer (pH 8.0, 0.1mol / L). The above-mentioned treated bacterial liquid was placed in ice water for ultrasonic crushing until clarification, the ultrasonic crushing conditions were: working for 2s, interval of 5s, ultrasonic power 500W. The crushed 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. Load t...

Embodiment 3

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

[0045] Using the pET30a(+)-Bmtyrc recombinant plasmid as a PCR template, conventional T7F / R as a universal primer (primer sequence: T7F: 5'-TAATACGACTCACTATAGGG-3', T7R: GCTAGTTATTGCTCAGCGG see SEQ ID NO.12 and 13) for the 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, it is guaranteed 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 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 1 minute, and extension at 72°C for 1.5 minutes, a total of 25 cycles; finally, extension at 72°C for 10 minutes....

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Abstract

The invention belongs to the technical field of enzyme engineering, and relates to a tyrosinase mutant and application thereof. According to the tyrosinase mutant, a plurality of amino acid sites are mutated in wild type tyrosinase with an amino acid sequence as shown in SEQ ID NO.1. According to the tyrosinase mutant and the method for preparing theaflavin by using the tyrosinase mutant, the mutant has higher specific activity than wild type tyrosinase, and has higher yield during catalytic preparation of theaflavin.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and relates to a tyrosinase mutant and its application in synthesizing theaflavins. Background technique [0002] In China, tea, as a national drink, has a long cultural and historical origin. According to the production and properties of tea, it can be divided into six major tea categories: black tea, green tea, green tea, yellow tea, black tea, and white tea. Black tea, as the second largest tea category in my country, is produced in a wide range of regions, including Fujian, Guangdong, There are more than ten provinces including Yunnan and Taiwan, and Yunnan and Fujian are the main planting areas. The most obvious feature that black tea is different 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. Studies in recent years have shown that black tea has many physiological functions beneficial to health, su...

Claims

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Application Information

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Patent Type & Authority Applications(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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