Ethyl carbamate hydrolase mutant and preparation method and application thereof
阅读说明：本技术 一种氨基甲酸乙酯水解酶突变体及其制备方法和应用 (Ethyl carbamate hydrolase mutant and preparation method and application thereof ) 是由 杨丽娟 张献 冯志平 张耀 刘君 赵金松 袁思棋 刘训 罗惠波 于 2021-10-15 设计创作，主要内容包括：本发明公开了一种氨基甲酸乙酯水解酶突变体及其制备方法和应用,属于基因工程和酶工程技术领域,本发明通过分析野生型氨基甲酸乙酯水解酶的氨基酸序列及建模后三维结构,将其194位的天冬酰胺突变为缬氨酸,得到稳定性提高的氨基甲酸乙酯水解酶突变体。本发明中氨基甲酸乙酯水解酶突变体N194V最适温度为45℃(野生型为40℃),且在40℃下的半衰期由野生型的32.09min提高到了210.04min,突变体N194V在35℃以上都比野生型有更好的耐受性。同时突变体N194V对于pH稳定性及乙醇的耐受性也有所提高。氨基甲酸乙酯水解酶突变体能更好的应用于降解食品及酒精饮料中的氨基甲酸乙酯。(The invention discloses an ethyl carbamate hydrolase mutant and a preparation method and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. The optimum temperature of the ethyl carbamate hydrolase mutant N194V is 45 ℃ (40 ℃ for wild type), the half-life period at 40 ℃ is improved from 32.09min of the wild type to 210.04min, and the mutant N194V has better tolerance than the wild type at the temperature of more than 35 ℃. Meanwhile, the mutant N194V has improved pH stability and ethanol tolerance. The ethyl carbamate hydrolase mutant can be better applied to degrading ethyl carbamate in food and alcoholic beverages.)
1. An ethyl carbamate hydrolase mutant, wherein asparagine at position 194 is mutated to valine relative to an ethyl carbamate hydrolase parent having an amino acid sequence shown in SEQ ID No. 1.
2. The method for producing the mutant ethyl carbamate hydrolase according to claim 1, comprising the steps of:
step (1): designing a site-directed mutation primer, and carrying out whole plasmid amplification by taking a vector carrying the coding gene of the ethyl carbamate hydrolase as a template to obtain a recombinant plasmid containing the mutant ethyl carbamate hydrolase;
step (2): and (2) treating the recombinant plasmid obtained in the step (1) by Dpn I enzyme, transferring the treated recombinant plasmid into E.coliBL21, performing induced expression, and purifying to obtain the ethyl carbamate hydrolase mutant.
3. Use of the mutant ethyl carbamate hydrolase according to claim 1 for degrading ethyl carbamate in food and/or fermented alcoholic beverages.
4. An acid-resistant fermented preparation characterized by using the ethyl carbamate hydrolase mutant according to claim 1 as a main ingredient.
5. An ethanol-resistant fermented preparation, characterized by using the ethyl carbamate hydrolase mutant according to claim 1 as a main ingredient.
6. A heat-resistant fermented preparation characterized by using the ethyl carbamate hydrolase mutant according to claim 1 as a main ingredient.
Technical Field
The invention relates to the technical field of genetic engineering and enzyme engineering, in particular to an ethyl carbamate hydrolase mutant and a preparation method and application thereof.
Background
Ethyl Carbamate (EC), which is present in various fermented foods and alcoholic beverages, is a substance having carcinogenicity. In 2007, EC was classified as a class 2A carcinogen by International Agency for Research on cancer iarc (the International Agency for Research on cancer). The major methods for eliminating EC currently include fermentation process optimization, physical adsorption, metabolic engineering, and bio-enzymatic methods, with bio-enzymatic methods being considered the most desirable method.
The Urethane Hydrolase (UH) can hydrolyze EC to ethanol, carbon dioxide and ammonia, and has potential application value. Since the enzyme needs better stability for industrial application, the enzymological properties of the purified urethane hydrolase in the former period of the invention show that the stability is poor at 40 deg.C or above or at pH 5. Therefore, the method for carrying out the fixed-point modification on the stability of the ethyl carbamate hydrolase to obtain the ethyl carbamate hydrolase mutant with improved stability and the preparation method and application thereof are very significant.
Disclosure of Invention
In view of the above disadvantages, the present invention aims to provide a mutant of ethyl carbamate hydrolase, a preparation method and applications thereof, which can effectively solve the problem of poor stability of the existing ethyl carbamate hydrolase. The amino acid sequence of the wild-type ethyl carbamate hydrolase is shown in SEQ ID No.1 by analyzing the ethyl carbamate hydrolase derived from Candida parapsilosis (China center for type culture Collection, with the preservation number of CCTCC AY 2017001), and on the basis, the asparagine at position 194 is mutated into valine to obtain the ethyl carbamate hydrolase mutant with improved stability.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an ethyl carbamate hydrolase mutant, wherein the 194 th asparagine of the mutant is mutated into valine relative to an ethyl carbamate hydrolase parent with an amino acid sequence shown in SEQ ID No. 1.
The invention also provides a preparation method of the ethyl carbamate hydrolase mutant, which comprises the following steps:
step (1): designing a site-directed mutation primer, and carrying out whole plasmid amplification by taking a vector carrying the coding gene of the ethyl carbamate hydrolase as a template to obtain a recombinant plasmid containing the mutant ethyl carbamate hydrolase;
step (2): and (2) treating the recombinant plasmid obtained in the step (1) with Dpn I enzyme, transferring the treated recombinant plasmid into E.coli BL21(DE3), performing induced expression, and purifying to obtain the ethyl carbamate hydrolase mutant.
The invention also provides application of the ethyl carbamate hydrolase mutant in degradation of ethyl carbamate in food and/or fermented alcoholic beverages.
An acid-resistant fermented preparation contains the above mutant of ethyl carbamate hydrolase as main ingredient.
It should be noted that the acid-resistant fermentation preparation provided by the invention means that the pH of the working environment is as low as 5.
An ethanol-resistant fermented preparation contains the above mutant of ethyl carbamate hydrolase as main ingredient.
It should be noted that the acid-resistant fermentation preparation provided by the invention refers to the working environment with ethanol concentration as high as 15 wt%.
A heat-resistant fermented preparation contains the above mutant of ethyl carbamate hydrolase as main ingredient.
It should be noted that the heat-resistant fermentation preparation provided by the invention means that the optimal working environment temperature is 45 ℃.
In summary, the invention has the following advantages:
1. the stability of the ethyl carbamate hydrolase mutant provided by the invention is improved, the optimum temperature of the mutant N194V is 45 ℃ (the wild type is 40 ℃), the half-life period at 40 ℃ is improved from 32.09min of the wild type to 210.04min, and the mutant N194V has better tolerance than the wild type at the temperature of more than 35 ℃. Meanwhile, the mutant N194V has improved pH stability and ethanol tolerance. The ethyl carbamate hydrolase mutant can be better applied to degrading ethyl carbamate in food and alcoholic beverages.
Drawings
FIG. 1: purifying with an affinity chromatography Ni-NTA column to obtain a pure enzyme SDS-PAGE electrophoresis picture, M: protein Marker; 1: wild-type WT; 2: mutant N194V.
FIG. 2: comparison of optimal temperatures before and after mutagenesis.
FIG. 3: influence of temperature before and after mutation on the thermostability of the wild enzyme and the mutant enzyme.
FIG. 4: comparison of optimal pH before and after mutation.
FIG. 5: the influence of pH on acid resistance of the wild enzyme and the mutant enzyme before and after mutation.
FIG. 6: ethanol tolerance effects on wild-type and mutant enzymes before and after mutation.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Thus, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
In this example, site-directed mutagenesis and expression of the urethane hydrolase gene were carried out. The amino acid at position 194 was selected for mutation by analyzing the sequence and structure of urethane hydrolase, and the mutant primers were designed on-line using PrimerX (Table 1, in which the sequences of N194-R, N194-F and N194V-F are shown as SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, respectively). Site-directed mutagenesis is carried out by using a whole plasmid PCR amplification technology by taking the recombinant plasmid pET-28a-cpUH as a template. The amplified recombinant plasmid is treated by Dpn I enzyme (acting for 1 hour at 37 ℃), transformed into E.coli DH5 alpha competence to obtain a positive transformant, and the extracted plasmid is sent to Beijing engine science and technology Limited company (Chengdu) for sequencing verification. Transformation of the correctly mutated plasmids into E.coli BL21(DE3), overnight culture and activation of E.coli BL21/pET-28a-cpUH and E.coli BL21/pET-28a-cpUH (N194V), inoculation of seed solutions at 2% inoculum size into fresh LB medium, cultivation at 37 ℃ and 180r/min to OD6000.6-0.8, adding IPTG with the final concentration of 0.25mmol/L, and carrying out induction culture at 25 ℃ for 8 h.
The cultured recombinant bacteria E.coli BL21/pET-28a-cpUH and E.coli BL21/pET-28a-cpUH (N194V) bacterial liquid are collected at 4 ℃ and 7000 r/min. After washing the cells 2 times with 20mmol/L PBS (pH 7.4), the cells were resuspended and sonicated in an ice bath. The disrupted bacterial solution was centrifuged at 7000r/min at 4 ℃ for 30min, and the supernatant (crude enzyme solution of wild enzyme and mutant N194V) was collected. Before purification, the nickel column was equilibrated with a nickel column equilibration buffer (PBS buffer containing 250mmol/L NaCl, 20mmol/L imidazole, pH 7.4), and then the elution of the protein was carried out using an elution buffer (PBS buffer containing 250mmol/L NaCl, 20mmol/L L, pH 7.4.4 imidazole, 100 mmol/L). The purified protein samples (wild enzyme and mutant enzyme N194V) were then subjected to SDS-PAGE using 5% concentrated gel and 10% separation gel, stained with Coomassie Brilliant blue R250 (FIG. 1); wherein, the sequence of the ethyl carbamate hydrolase mutant is shown as SEQ ID No. 2.
TABLE 1 Ethyl carbamate hydrolase mutant primer sequences (N194V)
Example 2
In this example, the enzyme activity was measured. The specific process is as follows: accurately sucking 200 mu L of enzyme solution (PBS solution as a control) into a colorimetric tube, adding 800 mu L of 3% EC solution, carrying out water bath at 40 ℃ for 15min, adding 1mL of terminator (10% trichloroacetic acid), and uniformly mixing to terminate the reaction. Then adding 1mL of developer I (15g of phenol, 0.625g of sodium nitroferricyanide and with the constant volume of ultrapure water of 250mL), uniformly mixing, adding 1mL of developer II (13.125g of NaOH and 7.5mL of NaClO solution and with the constant volume of ultrapure water of 250mL), uniformly mixing, carrying out heat preservation in a water bath at 40 ℃ for 20min, finally, with the ultrapure water of 10mL, measuring the absorbance and calculating the enzyme activity under 625 nm. Definition of enzyme activity: decomposition of the substrate EC per minute gave 1. mu. mol NH4 +The required amount of enzyme is 1 enzyme activity unit (U).
Example 3
In this example, the mutant enzyme (N194V, i.e., the mutant urethane hydrolase) was analyzed for heat resistance and half-life. The specific process is as follows: the purified wild enzyme and mutant enzyme (N194V) were reacted at 25, 30, 35, 40, 45, 50, and 55 ℃ respectively, and the optimum reaction temperature was measured. The optimum temperature of the mutant enzyme was increased from 40 ℃ (wild enzyme) to 45 ℃ (fig. 2). And (3) storing the two purified enzyme solutions at different temperatures for 30min, measuring corresponding enzyme activity, and calculating the relative enzyme activity by taking the initial enzyme activity of the enzyme at each temperature as 100%. The mutant enzyme (N194V) has better stability than the wild enzyme at the temperature of more than 35 ℃, and does not change greatly relative to the initial enzyme activity after being preserved for 30min at the temperature of 35-40 ℃, while the enzyme activity of the wild enzyme is reduced to about 50 percent of the initial enzyme activity after being preserved for 30min at the temperature of 40 ℃ (figure 3).
The purified wild enzyme and the mutant enzyme (N194V) thereof are placed at 40 ℃ for heat preservation, and sampling is carried out every 10min to determine the enzyme activity. From formula lnCt＝lnC0+kt，t1/2The half-life of the enzyme was calculated as ln2/k, where C0Is a firstStarter activity, CtThe enzyme activity corresponding to the time t. The half-life of the mutant enzyme (N194V) at 40 ℃ was 210.04min, which was 6.54 times that of the wild-type enzyme (32.09min) (Table 2). It was found that the mutant enzyme (N194V) was improved in thermostability to a large extent as compared with the wild enzyme.
TABLE 2 half-lives of the wild enzyme and the mutant enzyme (N1694V) at 40 ℃
Example 4
In this example, the acid resistance of the mutant enzyme (N194V) was analyzed. The specific process is as follows: and (3) reacting the purified wild enzyme and mutant enzyme (N194V) with a substrate EC in buffers with different pH values (3, 4, 5, 6, 7, 8, 9 and 10), and measuring the enzyme activities of the two enzymes under different pH conditions after the reaction is finished so as to determine the optimal reaction pH value. The optimum pH of the mutant enzyme (N194V) was decreased from 8 (wild enzyme) to 7, and the enzyme activity of the mutant enzyme (N194V) was improved compared to the wild enzyme under weakly acidic conditions (pH 5, 6) (fig. 4). And storing the purified enzyme solution in buffer solutions with different pH values (5, 6, 7, 8 and 9), placing the buffer solutions in a refrigerator at 4 ℃ for 4 hours, and calculating the relative enzyme activity corresponding to the pH value by taking the initial enzyme activity of each pH value as 100%. As a result, the mutant enzyme (N194V) was more tolerant than the wild-type enzyme at each pH and was 95% or more (FIG. 5).
Therefore, the acid resistance of the mutant enzyme N194V was also improved compared to the wild-type enzyme.
Example 5
In this example, ethanol tolerance analysis of the mutant enzyme was performed. The specific process is as follows: adding the purified wild enzyme and the mutant enzyme (N194V) into ethanol solutions with different concentrations, keeping the temperature at 37 ℃ for 1h, and determining the residual enzyme activity of the wild enzyme and the mutant enzyme, wherein the enzyme activity determined by the enzyme which is not treated by ethanol is 100%. The tolerance of the mutant enzyme N194V to ethanol is improved, and when the ethanol concentration is 15 wt%, more than 90% of enzyme activity is retained; when the ethanol concentration reaches 20 wt%, 18.82% of enzyme activity is still retained, and compared with the residual enzyme activity of the wild enzyme, the residual enzyme activity is 4.67%, which is improved by 14.15% (fig. 6).
The foregoing is merely exemplary and illustrative of the present invention and it is within the purview of one skilled in the art to modify or supplement the embodiments described or to substitute similar ones without the exercise of inventive faculty, and still fall within the scope of the claims.
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