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Thermophilic esterase AFEST mutant and screening method and application thereof

A technology for mutants and thermophilic esters, which is applied in the field of thermophilic esterase AFEST mutants and its screening, can solve the problems of directed evolution screening that cannot obtain good results, low positive rate, time-consuming and laborious, etc., to shorten the screening cycle and express The effect of high level and shortened screening efficiency

Active Publication Date: 2016-03-23
WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the very low positive rate in the random mutation library, conventional screening methods based on microwell plates or substrate plates often fail to achieve good results in directed evolution screening due to low throughput, time-consuming and laborious.

Method used

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  • Thermophilic esterase AFEST mutant and screening method and application thereof
  • Thermophilic esterase AFEST mutant and screening method and application thereof
  • Thermophilic esterase AFEST mutant and screening method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of embodiment 1 wild-type esterase AFEST gene

[0041] The wild-type esterase AFEST gene was synthesized by Beijing Huada Gene Company, and its sequence is SEQ ID NO.1. Through the upstream primer SEQIDNO.3: 5'-CCGCGCGGCAGC catATG CTTGATATGCCAATC-3' (underlined base is restriction endonuclease NdeI recognition site) and downstream primer SEQIDNO.4: 5'-GAGCTCGAATTC ggatcc CTAGTCGAACACAAGAAGAG-3' (the underlined base is the restriction endonuclease BamHI recognition site) amplifies the target gene. The PCR reaction uses Takara's PrimeSTARMax polymerase. The PCR reaction conditions are: 98°C for 2min, then 98°C for 10sec, 55°C 15sec at ℃, 10sec at 72℃, a total of 30 cycles; the last 10min at 72℃. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and a band with a size of 1 kb was obtained, which was consistent with the expected result. DpnI digests the template, recovers, purifies the target fragment, performs do...

Embodiment 2

[0042] Expression, purification and activity determination of embodiment 2AFEST

[0043] The engineered bacteria in the glycerol tube were inoculated into a 4 mL LB medium test tube containing 100 μg / mL Kan at a volume ratio of 1%, and cultured at 37° C. and 220 rpm for 12 hours. Transfer the 4mL bacterial liquid to a 1LLB medium shake flask containing 50μg / mLKan, culture at 37°C 220rpm for about 2.5h, make the OD600 reach about 0.8, add 0.1mMIPTG inducer, and induce culture at 25°C 200rpm for 12-16h. The Escherichia coli cell suspension harvested after fermentation is ultrasonically disrupted, and after one-step Ni-NTA affinity chromatography treatment, the target protein with a purity of more than 95% can be obtained. For the determination of esterase AFEST activity, refer to the literature ArchBiochemBiophys.2000, 373(1): 182-92.

Embodiment 3

[0044] The construction of the large-capacity random mutation library of embodiment 3 esterase AFEST

[0045] The mutation library of AFEST is constructed by error-prone PCR (ep-PCR), wherein the mutation rate is achieved by adjusting the concentration of manganese ions in the PCR system. The error-prone PCR system is: DreamTaq TM (0.05U / μL) and its buffer (Takara), dATP(250μM), dGTP(250μM), dCTP(1050μM), dTTP(1050μM), AFESTupper(0.4μM), AFESTlower(0.4μM), AFEST-pET-28a Plasmid (0.2ng / μL), manganese chloride (0.2-0.8mM). Wherein AFESTupper sequence SEQIDNO.3 is 5'-CCGCGCGGCAGC catATG CTTGATATGCCAATC-3', AFESTlower sequence SEQ ID NO.4 is 5'-GAGCTCGAATTC ggatcc CTAGTCGAACACAAGAAGAG-3'. Aliquot the PCR system into 25 μL tubes for error-prone PCR (95°C for 3min, 1cycle; 95°C for 15s / 55°C for 30s / 72°C for 1min, 30cycles; 72°C for 5min, 1cycle). The purified target fragment was double digested with NdeI and BamHIII, then cloned into the vector pET28a with T4 ligase, and then...

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Abstract

The invention discloses a thermophilic esterase AFEST mutant. An amino acid sequence of the thermophilic esterase AFEST mutant is different from an amino acid sequence SEQ ID NO.2 of wild type thermophilic esterase AFEST in 1-5 amino acid residues; the difference comprises replacement or deficiency or insertion of amino acid and is further shown in L47R or S111T or A166V or D175V. The invention further discloses a gene for coding the thermophilic esterase AFEST mutant, a recombinant vector containing the gene and an engineering bacterium containing the gene. The invention further discloses a screening method of the thermophilic esterase AFEST mutant and application of the thermophilic esterase AFEST mutant in hydrolysis of short-chain carboxylic ester substrates. According to the thermophilic esterase AFEST mutant, the hydrolytic activity on p-nitrophenol butyric ester is increased by 4 times or above, and the heat stability equivalent to that of the wild type thermophilic esterase AFEST is maintained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a thermophilic esterase AFEST mutant and its screening method and application. Background technique [0002] Esterase is a biocatalyst with a wide range of uses. It can not only catalyze the hydrolysis of various esters in the aqueous phase, but also catalyze the synthesis and transesterification of esters in the non-aqueous phase. It is often used in Detergent, paper industry, food, pharmaceutical industry and bio-energy, etc. However, since natural enzymes function in a relatively mild environment in the body, industrial applications require enzymes to be used in relatively harsh environments (such as high temperature, extreme pH, organic solvents, unnatural substrates, product inhibition, etc.) Therefore, natural enzymes often encounter problems such as poor stability and low catalytic efficiency in application. Therefore, enzymes with good stability and high catalytic efficienc...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N1/21C12N1/15C12N1/19C12N11/00C12P7/52C12P13/00C12R1/19C12R1/125C12R1/465C12R1/66C12R1/865
CPCC12N9/18C12N11/00C12P7/52C12P13/008C12Y301/01079
Inventor 杨广宇马富强谢渊刘新花
Owner WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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