Esterase mutant degrading ethyl carbamate and use thereof

Abstract

The present application relates to the technical field of genetic engineering and enzyme engineering, and particularly relates to an esterase mutant for degrading ethyl carbamate and application thereof, the esterase mutant has a sequence with site mutation of an amino acid sequence shown as SEQ ID NO. 1, the site mutation is any one or several site combination mutations of V129S, V161L or I229M, wherein the V129S site mutation is that valine at the 129th site is mutated into serine, the V161L site mutation is that valine at the 161st site is mutated into leucine, and the I229M site mutation is that isoleucine at the 229th site is mutated into methionine. The present application is mutated by a method of site-directed mutagenesis, so as to change the amino acid sequence, realize the change of protein structure and function, and finally obtain the esterase mutant V129S / I229M with the hydrolysis rate of ethyl carbamate in an acidic environment increased to 6.55 times, which has high industrial application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering and enzyme engineering, and particularly relates to an esterase mutant for degrading ethyl carbamate and application thereof. BACKGROUND

[0002] Ethyl carbamate (EC), also known as urethane or urethane, is a kind of ester toxic and harmful substance widely existing in fermented food, which has potential carcinogenicity to human body and is mainly generated in the storage and transportation process of fermented food. The substance was initially used as an anti-tumor drug to inhibit the growth of bacterial and mouse tumor cells. With the continuous progress of research, the carcinogenic mechanism of EC was reported, that is, the EC oxidized by cytochrome P450 can cause DNA chain damage, thereby triggering cancer. And when the EC in fermented food is studied, it is found that the presence of ethanol can further increase the carcinogenic risk of EC.

[0003] EC in fermented food is mainly formed by the reaction of amido-containing precursor substances (urea, carbamoyl phosphate, citrulline, diethyl pyrocarbonate, etc.) with ethanol. At present, 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 direct and high efficiency because it can directly hydrolyze EC or EC precursor substances. For example, urease can decompose the main precursor substance of EC, urea, which can effectively reduce the generation of EC. In the actual production process, the most commonly used method to reduce the content of EC in finished wine is to hydrolyze urea by using acid urease. However, since urea is not the only precursor substance of EC, it is difficult to completely eliminate the formation of EC by this method. 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 becomes a more promising method.

[0004] However, it is difficult to completely eliminate the formation of EC because the formation mechanism of EC is complex and it is difficult to inhibit multiple EC formation mechanisms, making it difficult to completely eliminate EC precursors and thus eliminate the formation of EC. In addition, it is difficult to remove EC after its formation because of its very stable structure. Therefore, using enzymatic degradation to degrade the formed EC in the finished product is a relatively efficient method. EC hydrolases mainly include amylases and esterases. Amylases hydrolyze EC by breaking the amide bond, and esterases hydrolyze EC by breaking the ester bond. However, the main precursor of EC, urea, also contains an amide bond, and the urea content in many alcoholic beverages is more than 100 times the EC content, making it difficult for amylases to be well applied. Esterases are more potential EC hydrolases because they do not compete with urea for substrates when hydrolyzing ester bonds. The problem is that the existing EC hydrolase library cannot meet the actual needs of EC biodegradation in food. On the one hand, there are very few EC hydrolases discovered, and on the other hand, EC hydrolases still have problems such as substrate competition with 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, the present application improves the acid tolerance and catalytic rate of EC hydrolytic esterases through molecular modification, with the aim of applying them in industrial production. SUMMARY

[0005] Therefore, the purpose of the present application is to provide an esterase mutant for degrading ethyl carbamate and its application. The hydrolytic esterase mutant has significantly improved acid tolerance and catalytic rate.

[0006] In order to achieve the above purpose, the present application provides an esterase mutant, which 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 combinations of V129S, V161L or I229M. The V129S site mutation is to mutate the valine at position 129 to serine, the V161L site mutation is to mutate the valine at position 161 to leucine, and the I229M site mutation is to mutate the isoleucine at position 229 to methionine.

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

[0008] The mutation combinations are as follows: V129S+V161L, V129S+I229M, V161L+I229M, V129S+V161L+I229M.

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

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

[0011] The recombinant plasmid is linked with any of the DNA molecules. Further, the vector of the recombinant plasmid includes 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 application also provides a recombinant host cell containing the recombinant plasmid, and the recombinant cell 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 application also provides the use of the esterase mutant, the gene, the recombinant plasmid or the recombinant host cell in degrading ethyl carbamate.

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

[0017] Further, the food includes alcoholic beverage, and the esterase can be used to degrade ethyl carbamate in the alcoholic beverage.

[0018] The esterase mutant or the expression system containing the esterase mutant is used for degradation.

[0019] Further, the catalytic reaction pH of the esterase mutant is 3.5-8.0, and the temperature is 20-80℃.

[0020] The esterase mutant provided by the present application can solve the problem of low activity of ethyl carbamate hydrolytic esterase in the acid environment, and the acid tolerance and catalytic rate are higher than those of the wild type. The relative enzyme activity of the mutants V129S, V161L and I229M is still maintained at more than 65% under the condition of pH 4.5; and the residual enzyme activity of the mutants V129S, V161L and I229M is still maintained at more than 40% after being stored for 6h under the condition of pH 4.0. In addition, the catalytic rate of the esterase mutant provided by the present application is also improved, and the catalytic rates of the 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 enhancement of acid tolerance and the improvement of catalytic rate are expected to be better applied in fermented food and fermented alcoholic beverage. The thermal stability of the mutant V161L is significantly increased, and the residual enzyme activity is still 86.5% after being stored for half an hour at 70℃; and the optimal reaction temperature of V161L is increased from 55℃ of the wild type to 65℃, which is expected to be applied in high-temperature environment. The present application is mutated by the method of site-directed mutation, so as to change the amino acid sequence, realize the change of protein structure and function, and finally obtain the esterase mutant V129S / I229M with the hydrolysis rate of ethyl carbamate in the acid environment increased by 6.55 times, which has high industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description only illustrate the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0022] Figure 1 For the SDS-PAGE diagram of the purified protein in the present application and the specific enzyme activity determination: (A) SDS-PAGE diagram of the protein (M is a standard molecular weight protein, and 1-7 lanes are Est8, V129S, V161L, I229M, V129S / I229M, V161L / I229M and V129S / V161L / I229M after purification, respectively; (B) specific enzyme activity determination of the purified enzyme;

[0023] Figure 2 For K m value fitting schematic diagram;

[0024] Figure 3 For the optimum pH and pH stability of the esterase mutants of the present application: (A) optimum pH; (B) pH stability;

[0025] Figure 4 For the optimum temperature and temperature stability of the esterase and its mutants of the present application: (A) optimum temperature; (B) temperature stability;

[0026] Figure 5 For the ethanol tolerance and ethanol stability of the esterase mutants of the present application, (A) ethanol tolerance; (B) ethanol stability. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present application more clear and apparent, the present application will be further described in detail in combination with specific embodiments.

[0028] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by those skilled in the art in the field to which the present application belongs. The words such as "comprise" or "include" and the like mean that the elements or objects before the words cover the elements or objects listed after the words and their equivalents, and do not exclude other elements or objects.

[0029] In the present application, the LB culture medium formula adopted is: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L; the TB culture medium formula adopted is: yeast extract 24 g / L, tryptone 12 g / L, 4 mL of glycerol, 2.313 g of potassium dihydrogen phosphate and 12.54 g of dipotassium hydrogen phosphate. (Solid culture medium additionally adds 15 g / L of agar powder)

[0030] The following commercial plasmids and E. coli are used for gene cloning and expression:

[0031] pET30a (Novagen, USA)

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

[0033] The method for determining the activity of the carbamate hydrolase enzyme: Take two 1.5 mL EP tubes, and add 50 μL of the diluted enzyme solution and 50 μL of the high-temperature 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 tube, and react in a 37℃ constant temperature water bath for 20 min. Then add 200 μL of the stop reagent (10% mass concentration of trichloroacetic acid) to each tube, mix well, and then add 200 μL of color developing reagent I (15 g of phenol and 0.625 g of nitrosoferricyanide sodium are dissolved in ultrapure water to a volume of 250 mL) and 200 μL of color developing reagent II (13.125 g of NaOH and 7.5 mL of NaClO are dissolved in ultrapure water to a volume of 250 mL), and shake vigorously. Then take out the tubes after further incubation in a 37℃ constant temperature water bath for 20 min, and measure the OD value at 625 nm, and convert the enzyme activity according to the standard curve prepared.

[0034] Definition of the unit of enzyme activity of the carbamate hydrolase: 1 μmol of NH4 + The required amount of enzyme is one unit of enzyme activity (U).

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

[0036] The template amino acid sequence of the present application is SEQ ID NO. 1. First, a mutation site is introduced into the esterase through 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] Wherein the site-directed mutation refers to introducing the desired change (usually a change characterized in a favorable direction) into the target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), including addition, deletion, point mutation, etc. of bases. The method of introducing site-directed mutation by whole plasmid PCR is simple and effective, and is a means currently used more. The principle is that a pair of primers containing the mutation site (forward and reverse), and the template plasmid are annealed and then extended with DNA polymerase to obtain a recombinant plasmid carrying the mutation site. The extension product is digested with Dpn I, and since the original template plasmid is derived from conventional E. coli, it is dam methylated and sensitive to Dpn I, so it is cut into pieces, while the in vitro synthesized plasmid with the mutated sequence is not methylated and is not cut, so it can be successfully transformed in the subsequent transformation, i.e. the mutant plasmid clone can be obtained. The mutant plasmid obtained by chemical transformation method is transformed into E. coli competent cells to obtain the mutant strain.

[0038] Example 1

[0039] Esterase mutation strategy

[0040] The Est8 protein structure simulated in this example is derived from the AlphaFold server (https: / / alphafoldserver.com / ), and the obtained protein model is subjected to molecular docking using Discovery Studio 4.5 software. In order to obtain an esterase mutant with improved catalytic rate in an acidic environment, all amino acid residues forming the substrate channel are selected for sequence analysis. After statistical analysis of the conservation and frequency of the selected amino acid residues, V129S, V161L and I229M are selected for site-directed mutation.

[0041] Preparation of esterase mutants

[0042] The selected mutation sites are designed for site-directed mutation primers, and the template is the pET-30a plasmid connected with the Est8 fragment. The mutation primers are used for amplification, and the primer sequences (SEQ ID NO. 3-8) are shown in Table 1. The amplified fragments are digested with Dpn I to prevent the unmutated plasmid from being indistinguishable from the mutated plasmid, forming false positive plasmids. The digested plasmid can be directly transformed into BL21(DE3) competent cells, and after incubation at 37°C for half an hour, it is plated on a kanamycin-containing plate, and positive transformants are selected. After picking a single colony from the plate, it is transferred to LB liquid medium and cultured for 12 hours, then stored in a glycerol tube at -80°C refrigerator, and samples are sent to Shanghai Bioengineering Co., Ltd. for sequencing verification.

[0043] Table 1 Primer sequences for constructing mutants

[0044]

[0045]

[0046] Note: The mutation site is underlined.

[0047] Culture and purification of esterase and its mutant enzymes

[0048] The correct strain with sequencing results was inoculated from the glycerol tube into LB liquid medium containing kanamycin and cultured at 37°C and 200 r / min for 12 h for activation of the strain. Then it was re-inoculated into LB liquid medium containing kanamycin and cultured at 37°C and 200 r / min for 12 h as a seed liquid for expressing protein. The seed liquid was inoculated into TB medium at a volume of 1% and cultured at 37°C and 200 r / min until the OD 600 value was 0.6-0.8, IPTG was added to a final concentration of 0.8 mmol / L, and the culture was incubated at 23°C and 200 r / min for 15 h. The recombinant bacterial liquid was centrifuged at 4°C and 8000 r / min for 10 min, and the bacterial cells were collected. The bacterial cells were washed with 20 mmol / L phosphate buffer at pH 7.0 and resuspended, and then ultrasonicated for half an hour at a power of 400 W, with 2 s of crushing and 3 s of stopping. The crushed bacterial liquid was centrifuged at 4°C and 10000 r / min for 30 min, and the supernatant was collected and used as the sample for loading after membrane filtration. During purification, the pipeline was first flushed with phosphate buffer containing 20 mmol / L imidazole and the nickel column was balanced, then the sample was loaded, and the protein was eluted with phosphate buffer containing 500 mmol / L imidazole. The eluate with enzyme activity was desalted using a desalting column, and the purified enzyme liquid was observed on a 10% SDS-PAGE gel to determine the apparent molecular weight and uniformity of the purified enzyme Figure 1 A), and the results showed that the enzyme was well purified. (The use of the purification column and the desalting column was performed according to the instructions on the Cytiva website)

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

[0050] Kinetic parameter determination of esterase and its mutant enzymes

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

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

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

[0054]

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

[0056] The enzyme activity of Est8 and its mutants was determined at pH 3.0-8.0 (20 mM citric acid-sodium phosphate dibasic), and the highest enzyme activity obtained was taken as 100%, and the enzyme activity measured under other pH conditions was taken as the percentage of the maximum enzyme activity. The enzyme activity determination results are shown in Figure 3As shown in Figure A, the optimal reaction pH for all mutants remains the same as that of the wild-type enzyme at pH 7.0. Furthermore, within the pH range of 5.0-8.0, both mutants and wild-types maintain more than 80% of their enzyme activity, demonstrating a relatively wide pH reaction range. Mutants I229M and V161L still maintain 78.9% and 75.6% of their relative enzyme activity at pH 4.0, respectively. This allows them to maintain high urethane degradation activity even in the acidic environments of most fermented foods (such as rice wine with a pH of 4.0-5.0).

[0057] To determine pH stability, the initial enzyme activity was set at 100%. Diluted pure enzymes were added to buffers of different pH values ​​(20 mmol / L citrate-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 incubated at 4°C for 6 hours. Residual enzyme activity was then measured. The enzyme activity results are shown below. Figure 3 As shown in B, under neutral conditions, both the esterase and its mutants can maintain high residual enzyme activity, especially the mutant V161L, which has the highest residual enzyme activity of 64.1% at pH 4. In addition, combined with the analysis of the relative enzyme activity of V161L retained at pH 4-6 in the optimal reaction pH determination, it has the best acid tolerance among all mutants. This makes it possible to use the esterase to reduce the EC content in acidic fermented foods.

[0058] Optimal temperature and temperature stability of esterases and their mutant enzymes

[0059] To determine the optimal reaction temperature of esterase, the enzyme activity of the purified enzyme solution was measured at different temperatures (20℃-80℃). 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 for EC hydrolytic esterase. The results showed ( Figure 4 A) The optimal reaction temperatures of the three single mutants are different. V129S has the same optimal temperature as the wild-type enzyme, 55℃, while the optimal reaction temperatures of V161L and I229M are increased to 65℃ and 60℃, respectively. Furthermore, the optimal reaction temperatures of the two-point mutant V129S / I229M and the three-point mutant V129S / V161L / I229M obtained by combining the three single mutants are lower than those of the wild-type enzyme, at 45℃. All six mutants maintain a relative enzyme activity of more than 40% in the range of 40℃-60℃, showing a wide 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 20-80°C for 1 h, and then rapidly heated or cooled to the corresponding optimal reaction temperature. The residual enzyme activity was determined at the optimal reaction temperature and pH 4.5, with the untreated enzyme activity defined as 100%. The residual enzyme activity of the mutants was calculated. Figure 4 B), the thermal stability of mutant V161L was significantly improved, and the residual enzyme activity was 86.5% after incubation at 70°C for half an hour. The addition of site 161 also significantly improved the thermal stability of mutant I229M. The residual enzyme activity of I229M was 25.8% at 70°C, while the relative enzyme activity of double mutant V161L / I229M was 65.8% under the same conditions, i.e., 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 diluted enzyme solution was added to a pH 4.5 citric acid-disodium hydrogen phosphate buffer containing 340 mM substrate and different volume fractions of ethanol (0%-40%) to determine the enzyme activity and investigate the effect of different volume fractions of ethanol solution on enzyme activity. The results of ethanol tolerance of the mutants showed that Figure 5 A), the six mutants had the same downward trend as the wild-type enzyme, but the relative enzyme activity of mutant V161L was significantly improved compared to WT under the conditions of 10%-30% ethanol concentration.

[0063] To determine the stability of esterase to ethanol, the esterase and its mutants were incubated at 37°C for 2 h under the above different concentrations of ethanol to investigate the stability of the esterase and its mutants under different ethanol and sodium chloride, with the untreated enzyme activity defined as 100%. In the ethanol stability determination Figure 5 B), the residual enzyme activity trends of all enzymes were nearly the same, and the mutation did not significantly affect the ethanol stability of the enzyme.

[0064] It should be understood by those of ordinary skill in the art that the above discussion of any of the embodiments is merely exemplary and is not intended to suggest that the scope of the application is limited to these examples; under the idea of the application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other changes of different aspects of the application as described above. In order to be brief, they are not provided in detail. Any omissions, modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. An esterase mutant, characterized in that, The esterase mutant is obtained by site mutation of the amino acid sequence shown in SEQ ID NO.

1. The site mutation is any one of V129S, V161L, and I229M, or a combination of V129S+I229M, V161L+I229M, and V129S+V161L+I229M. Specifically, the V129S site mutation is to mutate valine at position 129 to serine, the V161L site mutation is to mutate valine at position 161 to leucine, and the I229M site mutation is to mutate isoleucine at position 229 to methionine.

2. A gene encoding the esterase mutant of claim 1.

3. A recombinant plasmid containing the gene of claim 2.

4. A recombinant host cell, characterized in that, The recombinant host cell contains the recombinant plasmid of claim 3, and the host cell includes a prokaryotic cell or a eukaryotic cell.

5. The recombinant host cell according to claim 4, characterized in that, The prokaryotic cells are Escherichia coli BL21 cells or Escherichia coli DH5α cells, and the eukaryotic cells are yeast.

6. The use of the esterase mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, and the recombinant host cell of claim 4 or 5 in the degradation of ethyl carbamate.

7. The application according to claim 6, characterized in that, The application is the degradation of ethyl carbamate in the preparation of food.

8. The application according to claim 6, characterized in that, The catalytic reaction of the esterase mutant is carried out at a pH of 3.5-8.0 and a temperature of 20-80℃.

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