Esterase mutant for degrading ethyl carbamate and application thereof

Abstract

The invention relates to the technical field of gene engineering and enzyme engineering, in particular to an esterase mutant for degrading ethyl carbamate and application of the esterase mutant, the esterase mutant has an amino acid sequence as shown in SEQ ID NO.1 which is subjected to site mutation, the site mutation is any one or more site combined mutation of V129S, V161L or I229M, and the amino acid sequence of the esterase mutant is shown in SEQ ID NO.1. The V129S site mutation is that valine at the 129 site is mutated into serine, the V161L site mutation is that valine at the 161 site is mutated into leucine, and the I229M site mutation is that isoleucine at the 229 site is mutated into methionine. Mutation is carried out through a site-directed mutagenesis method, so that the amino acid sequence of the esterase mutant V129S / I229M is changed, the autoplasmic structure and function of eggs are changed, the esterase mutant V129S / I229M with the ethyl carbamate hydrolysis rate increased to 6.55 times in an acid environment is finally obtained, and the esterase mutant V129S / I229M has high industrial application value.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and enzyme engineering, and particularly relates to an esterase mutant for degrading ethyl carbamate and its application. Background Art

[0002] Ethyl carbamate (EC), also known as urethane, is an ester toxic and harmful substance widely present in fermented foods. It has potential carcinogenicity to humans and is mainly produced during the storage and transportation of fermented foods. This substance was initially used as an anti-tumor drug to inhibit the growth of bacteria and mouse tumor cells. With the continuous advancement of research, the carcinogenic mechanism of EC has been reported, that is, EC oxidized by cytochrome P450 will cause DNA strand damage, thereby triggering cancer. Moreover, when studying EC in fermented foods, it was found that the presence of ethanol will further increase its carcinogenic risk.

[0003] EC in fermented foods is mainly formed by the reaction of carbamyl precursor substances (urea, carbamyl phosphate, citrulline, diethyl pyrocarbonate, etc.) with ethanol. Currently, the elimination strategies of EC mainly include process optimization method, metabolic engineering method and enzyme method. Among them, the enzyme method has the characteristics of directness and high efficiency because it can directly hydrolyze EC or EC precursor substances. For example, urease can decompose urea, the main precursor substance of EC, and can effectively reduce the formation of EC. The most commonly used method to reduce the content of EC in finished wine in the actual production process is to use acidic urease to hydrolyze urea. However, since urea is not the only precursor substance for the formation of EC, this method is difficult to completely eradicate the formation of EC. Because EC hydrolase can degrade EC into ammonia, ethanol and carbon dioxide, it can effectively degrade EC. Therefore, using EC hydrolase to directly degrade EC in fermented products has become a more promising method.

[0004] However, since the EC formation mechanism is diverse and relatively complex, and it is difficult to inhibit multiple EC formation mechanisms simultaneously, it is hard to completely eradicate the EC precursor substances, thus making it difficult to completely eliminate the formation of EC. Moreover, once EC is formed, it is difficult to remove due to its very stable structure. Therefore, using enzymatic degradation to remove the already formed EC in the finished product is a relatively efficient method. EC hydrolases mainly include amidases and esterases. Amidases hydrolyze EC by breaking the amide bond, and esterases hydrolyze EC by breaking the ester bond. However, the main EC precursor substance, urea, also contains an amide bond, and in many alcoholic beverages, the urea content is more than a hundred times that of the EC content, resulting in poor application of amidases. Esterases, on the other hand, have become more promising EC hydrolases because hydrolyzing the ester bond does not form substrate competition with urea. The problem is that the currently existing EC hydrolase library far from meets the actual needs of EC biodegradation in food. On the one hand, very few EC hydrolases have been discovered. On the other hand, EC hydrolases still have problems such as substrate competition with the EC precursor substance urea, low activity in acidic or ethanol environments, weak affinity for EC, and poor catalytic efficiency, which further limits the application of EC hydrolases in actual production. Therefore, in the present invention, the EC hydrolase is modified by molecular means to improve its acid tolerance and catalytic rate in order to be applicable to industrial production. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide an esterase mutant for degrading ethyl carbamate and its application. The acid tolerance and catalytic rate of this hydrolytic esterase mutant are significantly improved.

[0006] Based on the above object, the present invention provides an esterase mutant. The esterase mutant has a sequence with site mutations in the amino acid sequence shown in SEQ ID NO.1. The site mutations are any one or several site combination mutations among V129S, V161L, or I229M. Among them, the V129S site mutation is the mutation of valine at position 129 to serine, the V161L site mutation is the mutation of valine at position 161 to leucine, and the I229M site mutation is the mutation of isoleucine at position 229 to methionine.

[0007] The esterase mutant has a homology of more than 99% with the esterase shown in SEQ ID NO.1 and has esterase catalytic activity.

[0008] Among them, the mutation combinations are as follows: V129S + V161L, V129S + I229M, V161L + I229M, V129S + V161L + I229M.

[0009] The present invention also provides a gene encoding the esterase mutant. This gene is a DNA molecule obtained by substitution, addition or deletion of bases in the sequence of SEQ ID NO.2, and this DNA molecule encodes any of the above esterase mutants.

[0010] The present invention also provides a recombinant plasmid containing the gene.

[0011] This recombinant plasmid is ligated with any of the above DNA molecules. Further, the vectors of the recombinant plasmid include pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 or pUC-19.

[0012] The present invention also provides a recombinant host cell, which contains the recombinant plasmid and is a non-plant cell.

[0013] Preferably, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0014] Preferably, the prokaryotic cell is Escherichia coli BL21 cell or Escherichia coli DH5α cell, and the eukaryotic cell is yeast.

[0015] The present invention also provides the application of the esterase mutant, the gene, the recombinant plasmid and the recombinant host cell in degrading ethyl carbamate.

[0016] Preferably, the application is for degrading ethyl carbamate in the preparation of food or alcoholic beverages.

[0017] Furthermore, the food includes alcoholic beverages, and this esterase can be used to degrade ethyl carbamate in alcoholic beverages.

[0018] The present invention uses the esterase mutant or the expression system containing the esterase mutant for degradation.

[0019] Furthermore, the pH of the catalytic reaction of the esterase mutant is 3.5 - 8.0, and the temperature is 20 - 80 °C

[0020] Advantages of the present invention: The esterase mutant provided by the present invention can solve the problem that the current ethyl carbamate-hydrolyzing esterase has low activity in an acidic environment, and its acid tolerance and catalytic rate are both improved compared with the wild type. Among them, mutants V129S, V161L, and I229M still maintain a relative enzyme activity of more than 65% under the condition of pH 4.5; after refrigeration at pH 4.0 for 6 h, the residual enzyme activities of mutants V129S, V161L, and I229M still remain above 40%. In addition, the catalytic rate of the esterase mutant provided by the present invention is also improved. Among them, the catalytic rates of mutants V129S / I229M, V161L / I229M, and V129S / V161L / I229M are increased from 161.46 / (s -1 ·mol / L -1 ) of the wild-type enzyme to 1057.69 / (s -1 ·mol / L -1 ), 740.64 / (s -1 ·mol / L -1 ), and 843.11 / (s -1 ·mol / L -1 ), respectively. The enhanced acid tolerance and the increased catalytic rate are expected to enable it to be better applied to fermented foods and fermented alcoholic beverages. The thermal stability of mutant V161L is significantly increased, and there is still 86.5% residual enzyme activity after incubation at 70 °C for half an hour; and the optimal reaction temperature of V161L is increased from 55 °C of the wild type to 65 °C, and it is expected to be applied in a high-temperature environment. The present invention performs mutations by site-directed mutagenesis to change its amino acid sequence, realize the change of the protein structure and function, and finally obtain the esterase mutant V129S / I229M with the hydrolysis rate of ethyl carbamate increased to 6.55 times in an acidic environment, which has high industrial application value. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 Purified protein SDS-PAGE diagram and specific enzyme activity determination for the present invention: (A) Protein SDS-PAGE diagram (M is the standard molecular weight protein, and lanes 1-7 are respectively purified Est8, V129S, V161L, I229M, V129S / I229M, V161L / I229M, and V129S / V161L / I229M; (B) Specific enzyme activity determination of the purified enzyme;

[0023] Figure 2 For the K m value fitting schematic diagram of Est8 and its mutants;

[0024] Figure 3 Schematic diagram of the optimal pH and pH stability of the esterase mutants of the present invention: (A) Optimal pH; (B) pH stability;

[0025] Figure 4 Optimal temperature and temperature stability of the esterase and its mutants of the present invention: (A) Optimal temperature; (B) Temperature stability;

[0026] Figure 5 Ethanol tolerance and ethanol stability of the esterase mutants of the present invention, (A) Ethanol tolerance; (B) Ethanol stability. Detailed implementation manners

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with specific embodiments.

[0028] It should be noted that unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those with ordinary skills in the field to which the present invention belongs. Words such as "including" or "comprising" mean that the elements or objects appearing before this word cover the elements or objects listed after this word and their equivalents, without excluding other elements or objects.

[0029] In the present invention, the formulation of the LB medium is: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L; the formulation of the TB medium is: yeast extract 24 g / L, tryptone 12 g / L, 4 mL glycerol, 2.313 g potassium dihydrogen phosphate, and 12.54 g dipotassium hydrogen phosphate. (For solid medium, an additional 15 g / L of agar powder is added)

[0030] The following commercialized plasmids and Escherichia coli are used for gene cloning and expression:

[0031] pET30a (Novagen, USA)

[0032] E. coli BL21(DE3) (Novagen, USA)

[0033] Method for determining the enzyme activity of ethyl carbamate hydrolase: Take two 1.5 mL EP tubes, and add 50 μL of the diluted enzyme solution and 50 μL of the heat-inactivated enzyme solution respectively. Then add 350 μL of 340 mM EC substrate solution (prepared with 20 mM, pH 7.0 phosphate buffer) to each of the two tubes. After reacting in a 37 °C constant temperature water bath for 20 min, add 200 μL of the terminator (10% mass concentration trichloroacetic acid) to each tube. After mixing, add 200 μL of color reagent I (15 g phenol and 0.625 g sodium nitroferricyanide made up to 250 mL with ultrapure water) and 200 μL of color reagent II (13.125 g NaOH and 7.5 mL NaClO made up to 250 mL with ultrapure water), shake vigorously, continue to incubate in a 37 °C constant temperature water bath for 20 min and then take out. Measure the OD value at 625 nm, and convert the enzyme activity according to the prepared standard curve.

[0034] Definition of the enzyme activity unit of ethyl carbamate hydrolase: Under normal pressure, in 20 mM citric acid - disodium hydrogen phosphate buffer at pH 4.5 and at 37 °C, the amount of enzyme required to degrade ethyl carbamate to generate 1 μmol of NH 4 + per minute is one enzyme activity unit (U).

[0035] The present invention improves the acid tolerance and catalytic rate of esterase through a rational design method.

[0036] The template amino acid sequence of the present invention is SEQ ID NO.1. First, mutation sites are introduced into the esterase by site-directed mutagenesis. The valine at position 129 is mutated to serine, the valine at position 161 is mutated to leucine, and the isoleucine at position 229 is mutated to methionine.

[0037] Among them, site-directed mutagenesis refers to introducing the required changes (usually changes representing the favorable direction), including base addition, deletion, point mutation, etc., into the target DNA fragment (which can be a genome or a plasmid) through methods such as polymerase chain reaction (PCR). The method of introducing site-directed mutagenesis using whole plasmid PCR is simple and effective, and is currently a widely used means. Its principle is that a pair of primers (forward and reverse) containing the mutation site anneal with the template plasmid, and then DNA polymerase is used for cyclic extension to obtain a recombinant plasmid carrying the mutation site. The extended product is digested with Dpn I enzyme. Since the original template plasmid is derived from conventional Escherichia coli and is methylated by dam, it is sensitive to Dpn I and is chopped up, while the in vitro synthesized plasmid with the mutant sequence is not cut because it has no methylation. Therefore, it can be successfully transformed in the subsequent transformation, and the clone of the mutant plasmid can be obtained. The obtained mutant plasmid is transformed into Escherichia coli competent cells by chemical transformation method to obtain the mutant strain.

[0038] Example 1

[0039] Esterase mutation strategy

[0040] In this example, the structure of the Est8 protein obtained by simulation is from the AlphaFold server (https: / / alphafoldserver.com / ), and the obtained protein model is used for molecular docking with Discovery Studio 4.5 software. To obtain an esterase mutant with an increased catalytic rate in an acidic environment, all amino acid residues forming the substrate channel were selected for sequence analysis. After statistical analysis of the conservation and frequency of the selected amino acid residues, V129S, V161L, and I229M were selected for site-directed mutagenesis.

[0041] Preparation of esterase mutant

[0042] Site-directed mutagenesis primers were designed for the selected mutation sites. The template was the pET-30a plasmid ligated with the Est8 fragment, and it was amplified using the mutant primers. The primer sequences (SEQ ID NO.3 - 8) are shown in Table 1. The amplified fragment was digested with Dpn I enzyme to digest the original plasmid template to prevent the undifferentiated non-mutated plasmid from forming a false positive plasmid with the mutated plasmid. The digested plasmid could be directly transformed into BL21(DE3) competent cells. After culturing at 37°C for half an hour, it was spread on a plate with kanamycin resistance to screen for positive transformants. Single colonies were picked from the plate and transferred to LB liquid medium for culturing for 12 h, then stored in a glycerol tube at -80°C in the refrigerator, and samples were taken and sent to Shanghai Bioengineering Co., Ltd. for sequencing verification.

[0043] Table 1 Primer sequences for constructing mutants

[0044]

[0045]

[0046] Note: The mutation sites are underlined.

[0047] Culture and Purification of Esterase and Its Mutant Enzymes

[0048] Inoculate the strain with correct sequencing results from the glycerol tube into LB liquid medium containing kanamycin resistance and culture it at 37 °C and 200 r / min for 12 h to activate the strain. Then re-transfer it to LB liquid medium containing kanamycin resistance and culture it at 37 °C and 200 r / min for 12 h as the seed liquid for expressing the protein. Transfer the seed liquid to TB medium at an inoculation amount of 1% by volume and culture it at 37 °C and 200 r / min until the OD 600 value reaches 0.6 - 0.8, add IPTG to a final concentration of 0.8 mmol / L, and culture it at 23 °C and 200 r / min for 15 h. Centrifuge the recombinant bacterial liquid at 4 °C and 8000 r / min for 10 min to collect the bacterial cells. Wash the bacterial cells with 20 mmol / L phosphate buffer at pH 7.0 and resuspend them, and ultrasonically disrupt them for half an hour at a power of 400 W with a condition of disrupting for 2 s and pausing for 3 s. Centrifuge the disrupted bacterial liquid at 4 °C and 10,000 r / min for 30 min and collect the supernatant, which is used as the sample for loading after passing through the membrane. During purification, first rinse the pipeline with phosphate buffer containing 20 mmol / L imidazole and equilibrate the nickel column, then load the sample, and elute the protein with a gradient of phosphate buffer containing 500 mmol / L imidazole. Desalt the eluate with enzyme activity using a desalting column, and observe the apparent molecular weight and homogeneity of the purified enzyme on a 10% SDS-PAGE gel ( Figure 1 A), and the results show that the enzyme has been well purified. (The use of the purification column and desalting column is operated according to the instructions on the Cytiva official website)

[0049] Determine the specific enzyme activity of the pure enzyme solutions of esterase and its mutants. The specific enzyme activity results of the wild-type esterase Est8 (patent number: CN2024101813667, the same below) and mutants are as Figure 1 shown in B. The specific enzyme activity of V161L / I229M is 216.3 U / mg, which is the highest among the mutants, while the specific enzyme activity of the wild-type enzyme is only 34.4 U / mg; the specific enzyme activities of all mutants V129S, V161L, I229M, V129S / I229M, V161L / I229M, and V129S / V161L / I229M are 1.36, 1.89, 5.06, 3.39, 6.28, and 4.83 times that of the wild-type enzyme, respectively.

[0050] Determination of Kinetic Parameters of Esterase and Its Mutant Enzymes

[0051] To investigate the affinity and catalytic activity of esterase for EC under acidic conditions, the purified esterase was reacted with EC at different final concentrations (10 - 1000 mmol / L), and the initial reaction rate of the enzyme was measured at pH 4.5. The GraphPad Prism software was used to analyze and fit the data, and the K m and V max (maximum velocity of enzymatic reaction) of the enzyme for EC were calculated. The formula for k cat (catalytic constant) is k cat = V max / [E], where [E] is the molar concentration of esterase.

[0052] The fitting results of the K m value are as shown in Figure 2 . The results of the kinetic parameter determination are shown in Table 2. The catalytic efficiency of all mutants was improved compared with the wild-type enzyme (WT). Among the single mutants, the K m value of V129S decreased to 196.13 ± 9.98 mmol / L, which was 23.45% lower than the K m value of the wild-type enzyme, indicating an improvement in its substrate affinity. After binding with the single mutant with the largest increase in catalytic rate, the double mutant V129S / I229M had the best substrate affinity and the largest catalytic rate. Its K m value was 120.44 ± 12.74 mmol / L, which was 52.99% lower than that of WT and the catalytic rate increased by 655.08%. V161L / I229M had the largest reaction rate among all mutants, which was 445.15 μmol / (min·mg), 557.06% of the maximum reaction rate of WT. This may be related to its highest relative enzyme activity among the mutants under the pH 4.5 environment. However, the poor substrate affinity of V161L / I229M led to a lower catalytic rate than that of V129S / I229M and V129S / V161L / I229M.

[0053] Table 2 Enzyme kinetic parameters of Est8 and its mutants

[0054]

[0055] Optimal pH and pH stability of esterase and its mutant enzymes

[0056] The enzyme activities of Est8 and its mutants were measured under the conditions of pH 3.0 - 8.0 (20 mM citric acid - disodium hydrogen phosphate) respectively. Taking the highest enzyme activity obtained from the measurement as 100%, the enzyme activities measured under other pH conditions were expressed as percentages of the maximum enzyme activity. The results of the enzyme activity measurement are as shown in Figure 3As shown in Figure A, the optimal reaction pH values of all mutants are still the same as that of the wild-type enzyme, which is pH 7.0. In the range of pH 5.0 - 8.0, both the mutants and the wild-type maintain more than 80% enzyme activity, showing a relatively broad pH reaction range; mutants I229M and V161L still maintain 78.9% and 75.6% relative enzyme activities under the condition of pH 4.0, which enables them to still have relatively high ethyl carbamate degradation activity in the acidic environment of most fermented foods (such as the pH value of 4.0 - 5.0 in yellow rice wine).

[0057] When measuring pH stability, with the initial enzyme activity as 100%, the diluted pure enzyme was added into buffer solutions with different pH values (20 mmol / L citric acid - disodium hydrogen phosphate buffer, pH 3.0 - 8.0; 20 mmol / L Tris - HCl buffer, pH 8.0 - 9.0; 20 mmol / L Gly - NaOH buffer, pH 9.0 - 10.0), and after refrigeration treatment at 4°C for 6 h, the residual enzyme activity was measured. The results of enzyme activity measurement are as Figure 3 shown in Figure B. In the neutral environment, both the esterase and its mutants can maintain relatively high residual enzyme activities. Especially for mutant V161L, its residual enzyme activity is up to 64.1% at pH 4.0. And combined with the analysis that V161L retains 75.6% - 89.4% relative enzyme activity at pH 4 - 6 in the optimal reaction pH measurement, it has the best acid tolerance among all mutants, which makes it possible to apply the esterase to reduce the content of EC in acidic fermented foods.

[0058] Optimal temperature and temperature stability of esterase and its mutant enzymes

[0059] To determine the optimal reaction temperature of the esterase, the purified enzyme solution was measured for enzyme activity at different temperatures (20°C - 80°C). The highest enzyme activity was defined as 100%, and the relative enzyme activity was calculated. The temperature corresponding to the maximum measured enzyme activity is the optimal reaction temperature of the EC - hydrolyzing esterase. The results show ( Figure 4 Figure A), the optimal reaction temperatures of the three single mutants are different. Among them, the optimal temperature of V129S is the same as that of the wild - type enzyme, which is 55°C, while the optimal reaction temperatures of V161L and I229M are increased to 65°C and 60°C. And the optimal reaction temperatures of the double - point mutant V129S / I229M and the triple - point mutant V129S / V161L / I229M obtained by combining mutations of the three single mutants are lower than that of the wild - type enzyme, which is 45°C; the six mutants maintain more than 40% relative enzyme activities at 40°C - 60°C, showing a relatively broad reaction temperature range and adaptability to different temperature environments.

[0060] To determine the thermal stability of the esterase, the purified enzyme solution was incubated at 20°C - 80°C for 1 h, and then quickly heated or cooled to the corresponding optimal reaction temperature. The residual enzyme activity was measured at the optimal reaction temperature and pH 4.5. The enzyme activity without incubation treatment was defined as 100%, and the remaining enzyme activity was calculated. In the results of the thermal stability determination of the mutants ( Figure 4 B), the thermal stability of the mutant V161L was significantly improved compared with that of the WT. After incubation at 70°C for half an hour, it still maintained 86.5% of the residual enzyme activity. In addition, the addition of the 161 site also significantly improved the thermal stability of the mutant I229M. The residual enzyme activity of I229M at 70°C was 25.8%, while the relative enzyme activity of the double mutant V161L / I229M in this environment was 65.8%, that is, the residual enzyme activity of V161L / I229M was 255% of that of I229M.

[0061] Ethanol tolerance of esterase and its mutant enzymes

[0062] The purified and diluted enzyme solution was added to the pH 4.5 citrate - disodium hydrogen phosphate buffer containing 340 mM substrate and different volume fractions of ethanol (0% - 40%) to measure the enzyme activity and investigate the effect of ethanol solutions with different volume fractions on the enzyme activity. In the results of the ethanol tolerance of the mutants ( Figure 5 A), the six mutants showed the same downward trend as the wild - type enzyme, but the relative enzyme activity of the mutant V161L was significantly improved compared with that of the WT in the environment with ethanol concentrations of 10% - 30%.

[0063] To determine the stability of the esterase to ethanol, it was incubated at 37°C for 2 h under the above - mentioned different ethanol concentrations to investigate the stability of the esterase and its mutants under different ethanol and sodium chloride conditions. The enzyme solution activity without pretreatment was taken as 100%. In the ethanol stability determination ( Figure 5 B), the trends of the residual enzyme activities of all enzymes were almost the same, and the mutation did not have a significant impact on the ethanol stability of the enzyme.

[0064] Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention is limited to these examples; under the concept of the present invention, the technical features between the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity. Any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An esterase mutant, characterized in that The esterase mutant has a sequence in which a site mutation occurs in the amino acid sequence as shown in SEQ ID NO.1, and the site mutation is any one of V129S, V161L or I229M, or a combination of several site mutations, wherein the V129S site mutation is a mutation of the valine at site 129 to serine, the V161L site mutation is a mutation of the valine at site 161 to leucine, and the I229M site mutation is a mutation of the isoleucine at site 229 to methionine.

2. A gene encoding the esterase mutant according to claim 1.

3. A recombinant plasmid containing the gene according to claim 2.

4. A recombinant host cell, characterized in that The recombinant host cell contains the recombinant plasmid according to claim 3, and the recombinant cell is a non-plant cell.

5. The recombinant host cell according to claim 4, characterized in that The host cell includes a prokaryotic cell or a eukaryotic cell.

6. The recombinant host cell according to claim 5, characterized in that The prokaryotic cell is an Escherichia coli BL21 cell or an Escherichia coli DH5α cell, and the eukaryotic cell is a yeast.

7. Use of the esterase mutant according to claim 1, the gene according to claim 2, the recombinant plasmid according to claim 3, and the recombinant host cell according to claims 4-6 in the degradation of ethyl carbamate.

8. The use according to claim 7, characterized in that: The application is the application of degrading ethyl carbamate in the preparation of food or alcoholic beverages.

9. The use according to claim 7, characterized in that: The degradation is carried out using the esterase mutant according to claim 1 or an expression system containing the esterase mutant.

10. The use according to any one of claims 7 to 9, characterized in that: The catalytic reaction pH of the esterase mutant is 3.5-8.0 and the temperature is 20-80°C.

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