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EstWY enzyme mutant having improved activity

A technology of mutants and activity, applied in the field of enzyme engineering, can solve the problems of poor enzyme activity of EstWY, and achieve the effect of simple culture conditions, good activity and high-efficiency expression

Active Publication Date: 2021-01-15
上海绅道生物科技有限公司
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  • Abstract
  • Description
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Problems solved by technology

[0003] In order to better apply Escherichia coli esterase (abbreviation: EstWY enzyme) to industrial processing and improve its physiological activity, the present invention adopts site-directed mutation to obtain a mutant enzyme with significantly improved activity, which solves the problem of poor activity of the existing EstWY enzyme The defects can not meet the needs of reagents, which laid the foundation for broadening the industrial application of EstWY enzymes

Method used

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  • EstWY enzyme mutant having improved activity
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  • EstWY enzyme mutant having improved activity

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Embodiment 1

[0027] Embodiment 1: the construction of single point EstWY enzyme mutant

[0028] The wild-type EstWY enzyme plasmid pET-28a was preserved in the laboratory, and a single-point EstWY enzyme mutant was constructed by the whole plasmid PCR method. The schematic diagram of the crystal structure of the EstWY enzyme ENND-TOP protein and the schematic diagram of the mutation site are as follows figure 1 shown. The details are as follows: using pET-28a as a template, the upstream and downstream primers of each mutation site are listed in Table 1, and are named in the format of "mutation site substitution amino acid". Using the high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase Kit, a round of PCR amplification was performed in order to obtain a gene recombinant plasmid containing the mutant. The reaction system is shown in Table 2. PCR conditions: pre-denaturation: 95°C for 3min; denaturation: 98°C for 10s; annealing: 58°C for 10s; extension: 72°C for 5min; cycle 35 times; fu...

Embodiment 2

[0034] Embodiment 2: the construction of multi-point EstWY enzyme mutant

[0035] In order to further analyze the effect of different amino acid species at each site on the catalytic properties of the enzyme, referring to the method of site-directed mutagenesis, the whole plasmid PCR technology is still used to obtain the saturated mutation library gene. The details are as follows: Use the high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase Kit, carry out multiple rounds of PCR amplification in order to obtain gene recombinant plasmids containing mutants. The reaction system, PCR conditions and transformation conditions are the same as those for site-directed mutagenesis.

Embodiment 3

[0036] Embodiment 3: construction of mutant engineered bacteria

[0037] Refer to the instructions of the Super Competent Kit to construct engineering bacteria. The specific operation is as follows. Firstly, confirm that E.coliBL21(DE3) cannot grow under Kan resistance; secondly, streak and activate the E.coli BL21(DE3); thirdly, take a single colony and add it to LB culture without resistance culture medium until the OD600 is between 0.6 and 0.8, and prepare competent cells with the kit’s own solution; the fourth step is to transform and spread on the LB solid medium plate containing Kan resistance, and cultivate for 16 hours; finally, pick For 3 single colonies, the target gene was amplified by bacterial liquid PCR, and the target band was identified by agarose gel electrophoresis, and then sent to Suzhou Jinweizhi for sequencing to confirm the engineering bacteria.

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Abstract

The invention discloses an EstWY enzyme mutant having improved activity, and belongs to the technical field of enzyme engineering. An EstWY enzyme sequence is analyzed by using a consensus method without phylogenetic prejudice to obtain single-point mutants having remarkably improved activity through screening, and site-specific mutagenesis is performed on the single-point mutants to obtain mutantY179E / V190D, T196R / L202H and V190D / A193H / T196R enzymes having improved activity, which are 3 times higher than wild enzymes. The EstWY enzyme mutant provided by the invention is better in activity and shows excellent catalytic activity when creatine is catalyzed to generate fatty acids and alcohols at a relatively high temperature, besides, the constructed EstWY enzyme genetically engineered bacterium can efficiently express the EstWY enzyme mutant, and the EstWY enzyme genetically engineered bacterium has the advantages of simple culture conditions, short culture period, convenience in purification of expression products and the like.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, in particular to an EstWY enzyme mutant with improved activity. Background technique [0002] Esterase is an enzyme system that catalyzes the hydrolysis of ester compounds, and its function is to hydrolyze aliphatic esters and aromatic esters. With the participation of water molecules, esters are hydrolyzed into acids and alcohols through hydrolysis. The reaction formula is as follows: R-COOR / (Ester)+H 2 O (water)====RCOOH (fatty acid)+R / OH (alcohol). It widely exists in animals, plants and microorganisms. Among them, animal pancreas esterase and microbial esterase are the main sources of esterase. Due to the abundant microbial resources and the advantages of convenient industrial production by using microbial fermentation to produce enzymes, esterases are widely used in fields such as agriculture, food brewing, pharmaceutical chemistry, sewage treatment and bioremediation. ...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N1/21C12N1/19C12R1/19C12R1/125C12R1/645
CPCC12N9/18Y02A50/30
Inventor 杨广宇郭天杰马富强李长龙秦朕龙
Owner 上海绅道生物科技有限公司
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