A kind of thermophilic esterase afest mutant and its screening method and application

A mutant and thermophilic ester technology, which is applied in the field of thermophilic esterase AFEST mutants and their screening, can solve the problems that directed evolution screening cannot obtain good results, is time-consuming and labor-intensive, and has low positive rate, and can shorten the screening period and shorten the Screening efficiency, high expression level effect

Active Publication Date: 2019-01-01
WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
View PDF0 Cites 1 Cited by
  • Summary
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of thermophilic esterase afest mutant and its screening method and application
  • A kind of thermophilic esterase afest mutant and its screening method and application
  • A kind of thermophilic esterase afest mutant and its screening method and application

Examples

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 SEQ ID NO.3: 5'-CCGCGCGGCAGC catATG CTTGATATGCCAATC-3' (underlined base is restriction endonuclease NdeI recognition site) and downstream primer SEQ ID NO.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 PrimeSTAR Max polymerase. The PCR reaction conditions are: 98°C for 2min, then 98°C for 10sec, 55°C for 15sec, 72°C for 10sec, a total of 30 cycles; finally 72°C for 10min. 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, perf...

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 220 rpm at 37°C for 12 hours. Transfer the 4mL bacterial liquid to a 1L LB medium shake flask containing 50μg / mL Kan, culture at 220rpm at 37°C for about 2.5h, make the OD600 reach about 0.8, add 0.1mM IPTG inducer, and induce culture at 25°C at 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 Arch Biochem Biophys.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), AFEST upper(0.4μM), AFEST lower(0.4μM), AFEST-pET -28a plasmid (0.2ng / μL), manganese chloride (0.2-0.8mM). Wherein AFESTupper sequence SEQ ID NO.3 is 5'-CCGCGCGGCAGC catATG CTTGATATGCCAATC-3', AFEST lower 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, an...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
Login to view more

Abstract

The invention discloses a thermophilic esterase AFEST mutant, the amino acid sequence of the thermophilic esterase AFEST mutant has 1-5 amino acid residues compared with the amino acid sequence SEQ ID NO.2 of the wild-type thermophilic esterase AFEST Different; said differences include substitutions, deletions or insertions of amino acids. Further, the difference is reflected in L47R, S 111T, A166V or D175V. The invention also protects a gene encoding the AFEST mutant of the thermophilic esterase, a recombinant vector containing the gene, and an engineering bacterium containing the gene. The invention also discloses a screening method of the thermophilic esterase AFEST mutant and its application in hydrolyzing short-chain carboxylate substrates. The hydrolysis activity of the thermophilic esterase AFEST mutant of the present invention to p-nitrophenol butyrate is increased by more than 4 times, and the thermostability 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap