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Ethyl carbamate hydrolase mutants with improved 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: 2018-08-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 hydrolase mutants with improved thermostability
  • Ethyl carbamate hydrolase mutants with improved thermostability
  • Ethyl carbamate hydrolase mutants with improved thermostability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Example 1 Preparation of urethane hydrolase mutants

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

Embodiment 2

[0015] Example 2 Enzyme activity assay method

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

Embodiment 3

[0017] Example 3 Mutation improves the thermostability and half-life of urethane 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 both wild enzyme and mutant was 30℃, but the stability of mutant Q328C, Q328R and Q328V was better than that of wild enzyme above 30℃. After incubation at 37°C for 1 time, the residual enzyme activities of Q328C, Q328R, Q328V and UH were 76.9%, 60.8%, 75.8% and 51.9%, respectively, which were 24.0%, 8.9% and 23.9% higher than the original enzyme. At 40°C, the residual enzyme activities of the mutants were increased by 31.7%, 19.7% and 28.9% compared with the original enzyme, respectively ( figure 1 ).

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

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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 carbamate hydrolase mutant with improved thermal stability, and belongs to the technical field of genetic engineering and enzyme engineering. Background technique [0002] Ethyl carbamate (EC) is a fermented food (such as soy sauce, fermented bean curd, kimchi) and alcoholic beverages (wine, rice wine, liquor) produced during the production and storage process, which is potentially carcinogenic and genotoxic to humans. traces of harmful substances. Because the formation mechanism of EC is complex, and once it is stable, 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 biological enzymatic method. Urethane hydrolase (Urethanase, UH) can degrade EC to generate harmless ethanol and carbon dioxide, which has potential industrial application va...

Claims

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Application Information

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Patent Type & Authority Patents(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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