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Ethyl carbamate hydrolytic enzyme mutants capable of improving thermostability

A technology of urethane and thermostability, which is applied in the field of genetic engineering and enzyme engineering, and can solve problems such as non-elimination and complex formation mechanism

Active Publication Date: 2016-03-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the complex formation mechanism of EC and its stable nature, once it is generated, it is difficult to remove it by physical or chemical methods. At present, there is no effective method to eliminate EC in different fermentation products.

Method used

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  • Ethyl carbamate hydrolytic enzyme mutants capable of improving thermostability
  • Ethyl carbamate hydrolytic enzyme mutants capable of improving thermostability
  • Ethyl carbamate hydrolytic enzyme mutants capable of improving thermostability

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Effect test

Embodiment 1

[0010] The preparation of embodiment 1 ethyl carbamate hydrolase mutant

[0011] By analyzing the structure of ethyl carbamate hydrolase, we chose to mutate the 328th amino acid, and designed corresponding site-directed mutagenesis primers (Table 1). Using the recombinant plasmid pET20b-UH as a template, PCR enzyme was used to amplify the recombinant plasmid pET20b-UH with mutant primers. The amplified fragments were recovered and purified using a gel recovery kit. Phosphorylate the purified fragment with phosphorylase. The phosphorylated fragments were ligated with ligase, transformed into Escherichia coli BL21(DE3) competent, positive transformants were screened, plasmids were extracted, identified by enzyme digestion, and sent to Shanghai Bioengineering Co., Ltd. for sequencing verification. The Escherichia coli recombinant engineered bacteria containing the correct sequenced plasmid were inoculated into LB medium and cultured overnight at 37°C and 220r / min. The seed sol...

Embodiment 2

[0015] Embodiment 2 enzyme activity assay method

[0016] Take 1mL of enzyme solution and 1mL of ultrapure water (control), add 1mL of 3% EC solution each, react in a constant temperature water bath at 37°C for 15min, then add 1mL of 10% trichloroacetic acid to terminate the reaction. After the reaction is terminated, add 1mL color developer I (15g phenol and 0.625g sodium nitroferricyanide to 250mL) and 1mL color developer II (13.125gNaOH and 7.5mL sodium hypochlorite to 250mL), mix well Incubate in a water bath at 37°C for 20 min. After the reaction, dilute to 10 mL with ultrapure water, and measure the absorbance at 625 nm. Definition of enzyme activity: under the conditions of normal pressure, 37°C, and pH 7.0, the amount of enzyme required to decompose EC to produce 1 μmol ammonia in 1 minute is one enzyme activity unit (U).

Embodiment 3

[0017] Embodiment 3 Mutation improves heat stability and half-life of ethyl carbamate hydrolase

[0018] The purified wild enzyme and its mutants were subjected to enzymatic reactions at different temperatures (20° C. to 65° C., with an interval of 5° C.), and the optimum reaction temperature was determined. The optimum reaction temperature of the wild enzyme and the mutant was 30°C, but the stability of the mutants Q328C, Q328R, and Q328V was better than that of the wild enzyme above 30°C. After incubation at 37°C for 1 hour, the residual enzyme activities of Q328C, Q328R, Q328V and UH were 76.9%, 60.8%, 75.8% and 51.9%, which were 24.0%, 8.9% and 23.9% higher than the original enzymes, respectively. At 40°C, the residual enzyme activities of the mutants were 31.7%, 19.7% and 28.9% higher than the original enzyme, respectively ( figure 1 ).

[0019] The purified wild enzyme and its mutants were incubated at 40°C, and samples were taken at regular intervals to determine the ...

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Abstract

The invention discloses ethyl carbamate hydrolytic enzyme mutants capable of improving the thermostability, and belongs to the technical field of gene engineering and enzyme engineering. By means of a molecular technical means, the ethyl carbamate hydrolytic enzyme mutants capable of improving the thermostability are obtained, and the half-life periods of the ethyl carbamate hydrolytic enzyme mutants Q328C and Q328V are increased by 7.46 times and 1.96 times respectively compared with a native enzyme. The mutants Q328C, Q328R and Q328 V all have better tolerance at 30 DEG C or above than the native enzyme. In addition, the tolerance to ethyl alcohol and the tolerance to acid of the mutant Q328C are improved.

Description

technical field [0001] The invention relates to a mutant of ethyl carbamate hydrolase with improved thermostability, and belongs to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Ethylcarbamate (EC) is a potential carcinogenic and genotoxic compound produced during the production and storage of fermented foods (such as soy sauce, fermented bean curd, pickles) and alcoholic beverages (wine, rice wine, liquor) Trace amounts of harmful substances. Due to the complex formation mechanism of EC and its stable nature, once it is formed, it is difficult to remove it by physical or chemical methods. At present, there is no effective method to eliminate EC in different fermentation products. It is a relatively safe and effective method to remove EC in finished products by using biological enzymatic method. Urethanase (UH) can degrade EC to generate harmless ethanol and carbon dioxide, which has potential industrial application value...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12H1/15
CPCC12H1/14C12N9/18
Inventor 方芳刘晓慧吕思熠陈坚堵国成
Owner JIANGNAN UNIV
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