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Ethyl carbamate hydrolase mutant and application thereof

A kind of urethane and hydrolase technology, applied in the biological field, can solve the problem of low applicability and so on

Active Publication Date: 2021-02-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because ethyl carbamate hydrolase is mainly used in fermented wine, but the alcohol concentration of traditional fermented wine is about 20%, it is acidic; therefore, the applicability of currently screened ethyl carbamate hydrolase is not high

Method used

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  • Ethyl carbamate hydrolase mutant and application thereof
  • Ethyl carbamate hydrolase mutant and application thereof
  • Ethyl carbamate hydrolase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Agrobacterium tumefaciens d 3 Heterologous expression of amidase

[0021] (1) Use primer sequence D 3 -F,D 3 -R (Table 1) amplifies the amidase gene of Agrobacterium tumefaciens d3 (entrusted to Qingke Biosynthesis, the gene sequence is GenBank: AF315580, the amino acid sequence is shown in SEQ ID No.1), with primers pET30-F, pET30 -R amplification to obtain a linearized vector. Then use the ClonExpress II one-step cloning kit (purchased from Novizyme, Cat. No. C112-02) to connect the gene fragment between the NdeI and HindIII sites of the vector. The constructed plasmid was named pET-30a(+) / AtUTNase. The gene on the constructed plasmid has a His-tag for easy purification.

[0022] Table 1 Primers

[0023]

[0024]

[0025] (2) Transform the plasmid pET-30a(+) / AtUTNase into Escherichia coli BL21(DE3), inoculate the Escherichia coli BL21(DE3) carrying pET-30a(+) / AtUTNase into a test tube containing kanamycin and cultivate overnight , transferred t...

Embodiment 2

[0027] Embodiment 2: the enzyme activity detection method of ethyl carbamate hydrolase

[0028] The principle of the method for detecting the enzyme activity of ethyl carbamate hydrolase is: under certain conditions, ethyl carbamate hydrolase decomposes ethyl carbamate to generate ethanol, ammonia and carbon dioxide. Ammonia reacts with phenol-sodium hypochlorite to form indophenol blue, which is blue, and its absorbance value is measured at 625nm with a spectrophotometer to analyze the amount of ammonia generated, so as to obtain the enzyme activity of ethyl carbamate hydrolase. Definition of enzyme activity: 1 μmol of ammonia produced by decomposing the substrate per minute under normal pressure and 37°C is an enzyme activity unit.

[0029] (1) Preparation of enzyme activity assay reagents:

[0030] Color developer I: Weigh 15g of phenol and 0.625g of sodium nitroferricyanide and dilute to 250mL with ultrapure water.

[0031] Chromogen II: Weigh 13.125g of sodium hydroxide...

Embodiment 3

[0039] Embodiment 3: the purification of amidase

[0040] (1) Prepare the solution:

[0041] Binding Buffer: Weigh 1.7g of imidazole and 14.6g of NaCl in a beaker, add 50mL of 0.2M NaCl 2 HPO 4 -NaHPO 4 Buffer, use phosphoric acid to adjust the pH, and finally dilute to 500mL with deionized water.

[0042] Elution Buffer: weigh imidazole 5.1, 5.97, 6.8, 8.5, 9.35, 10.2, 17g and NaCl 14.6g in a beaker, add 50mL 0.2M Na 2 HPO 4 -NaHPO 4 For the buffer solution, use phosphoric acid to adjust the pH, and finally use deionized water to adjust the volume to 500mL, so as to prepare elution buffer solutions with imidazole concentrations of 150mM, 175mM, 200mM, 250mM, 275mM, 300mM, and 500mM, respectively.

[0043] (2) Operation process:

[0044] Pretreatment: 10 times column volume ddH 2 O washing nickel column, 10 times of column volume 50mM imidazole buffer equilibrium nickel column;

[0045] Adding samples: Repeat loading of the broken cell solution five times;

[0046] W...

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Abstract

The invention discloses an ethyl carbamate hydrolase mutant and application thereof. The ethyl carbamate hydrolase mutant is obtained by mutation of an amidase gene of Agrobacterium tumefaciens d3, the amino acid sequence of wild amidase is shown as SEQ ID No.1, and the ethyl carbamate hydrolase mutant is obtained by mutation of at least one site of R94P, I97L, S177C or G195A of the wild amidase.The amidase from the Agrobacterium tumefaciens d3 is obtained by screening, the residual enzyme activity is higher than 90% when the ethanol concentration is between 0% and 20%, the enzyme activity isquickly reduced when the ethanol concentration is higher than 25% , and the amidase shows excellent tolerance to low-concentration ethanol. Through semi-rational transformation, a mutant library with21 point mutations is constructed, four mutation sites (R94P, I97L, S177C and G195A) with improved enzyme activity are screened from the mutant library, and the enzyme activity of a combined mutant strain I97L / G195A is improved by 5.2 times and is improved to 2442U / L from original 395U / L.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant of ethyl carbamate hydrolase and its application. Background technique [0002] Ethyl carbamate (Ethyl carbamate; C 2 h 5 OCONH 2 ), referred to as EC. Urethane is an important raw material in chemical production and has a wide range of uses. It has the effect of anesthesia and is used as a sedative and palliative hypnotic in medicine. Urethane is also widely found in traditional fermented foods such as kimchi, soy sauce, vinegar, and alcoholic beverages such as sake, wine, and rice wine. In 1943, the potential carcinogenicity of EC was discovered by Nettleship et al. In 1974, the International Agency for Research on Cancer (IARC) classified EC as a 2B carcinogen. In 2007, IARC officially raised the carcinogenicity level of EC from 2B to 2A. Experiments have proved that urethane is carcinogenic to all experimental animals and has potential carcinogenicity to humans. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12P7/06
CPCC12N9/80C12P7/06C12Y305/01075Y02E50/10
Inventor 吴绵斌康婷婷杨祎林建平杨立荣
Owner ZHEJIANG UNIV
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