A multi-tolerant amidase capable of degrading carbamate and amide compounds

Abstract

This invention discloses an amidase with multiple tolerances capable of degrading carbamates and amides. This enzyme can efficiently degrade carbamates and amides, exhibiting extremely high salt and acid tolerance. This invention successfully achieved efficient expression of the amidase gene and purification of the enzyme protein using an *E. coli* expression system, with a specific enzyme activity as high as 397.5 U / mg. Salt significantly promotes the enzyme activity at NaCl concentrations not exceeding 5% (w / v), maintaining 75% or more of the enzyme activity even at NaCl concentrations not exceeding 18% (w / v); it retains 40% of its activity at pH 4.5, and maintains approximately 43%-85% of its activity after 6 hours at pH 4.0-5.0, demonstrating strong acid tolerance. It also exhibits some ethanol tolerance. The amidase prepared by this invention lays the foundation for eliminating impurities in high-salt fermented foods and low-alcohol beverages, offering significant economic and social benefits.

Description

Technical Field

[0001] This invention relates to an amidase with multiple tolerances that can degrade carbamates and amides, belonging to the field of biotechnology. Background Technology

[0002] Ethyl carbamate (EC), also known as urethane, is a harmful nitrogenous metabolite commonly found in fermented foods and alcoholic beverages. It possesses genotoxicity and strong carcinogenicity, and is widely present in traditional fermented foods and alcoholic beverages such as rice wine, baijiu, wine, sake, and soy sauce. In 1943, Nettleship first confirmed the carcinogenicity of EC. Subsequent studies have shown that EC can induce skin cancer, lymphoma, and liver cancer in various experimental animals. In 2007, the International Agency for Research on Cancer (IARC) classified it as a Group 2A carcinogen, indicating that it is likely carcinogenic to humans. EC formation is related to precursors such as urea and ethanol during fermentation, and its content is influenced by various factors, including fermentation conditions, storage time, and temperature. To protect consumer health, many countries (such as the United States and Canada) have set strict limits on EC content in food and beverages. Methods to reduce EC content include process optimization, metabolic engineering of fermentation strains, and enzymatic degradation. Among these methods, enzymatic degradation directly breaks down ethyl carbamate (EC) into non-toxic ethanol, carbon dioxide, and ammonia using specific enzymes such as EC hydrolase. Without affecting the flavor of traditional fermented foods, enzymatic degradation can efficiently degrade ethyl carbamate formed through all pathways. Theoretically, compared to other methods, this is the most promising approach to completely solve the food safety problems caused by EC in traditional fermented foods.

[0003] However, currently there are no effective enzymes for the direct degradation of EC in traditional fermented foods (such as soy sauce). This is mainly due to significant shortcomings in the biochemical characteristics of the EC hydrolases discovered so far: First, most traditional fermented foods are acidic, and the ethyl carbamate hydrolases discovered so far exhibit poor acid tolerance, with low activity and extremely poor stability at pH 3-5, severely limiting their application in traditional fermented foods. Second, the EC hydrolases discovered so far exhibit poor salt tolerance, with extremely low activity or even loss of activity in environments containing high salt concentrations (NaCl, 18% w / v), thus limiting their application in high-salt fermented foods such as soy sauce. Third, the EC hydrolases discovered so far exhibit poor alcohol tolerance, with low activity and poor stability in the presence of ethanol (10% v / v). Therefore, screening for EC hydrolases with high activity and stability in acidic environments is key to solving the problem of EC degradation in traditional fermented foods. Meanwhile, if the selected enzymes also possess high EC degradation activity and stability in high-salt or ethanol-containing environments, their application in high-salt traditional fermented foods or alcoholic beverages will be enhanced.

[0004] Acrylamide is an organic compound classified as a Group 2A "probable human carcinogen" by the International Agency for Research on Cancer (IARC). It is primarily found in heat-processed foods such as fried and baked goods, including French fries, bread, biscuits, and instant coffee. It is produced through the Maillard reaction during the high-temperature heating of foods rich in carbohydrates and amino acids, primarily via the asparagine and acrolein pathways. Due to its neurotoxicity, reproductive toxicity, and genotoxicity, and its potential link to cancer, it is necessary to reduce its levels. Enzymatic degradation of acrylamide offers advantages such as high efficiency, specificity, and mild conditions, reducing acrylamide content without significantly affecting food quality, making it a relatively ideal degradation method. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes an amide with multiple tolerances that can degrade ethyl carbamate and acrylamide, and its applications, to solve the problems of low activity and poor stability of ethyl carbamate hydrolase under acidic, high-salt, and ethanol-containing conditions. This provides a new method for solving the degradation of EC in traditional fermented foods and the reduction of acrylamide in heat-processed foods such as fried and baked foods.

[0006] The first object of the present invention is to provide an amidase, the gene sequence encoding said amidase being shown in SEQ ID NO.2.

[0007] A second objective of the present invention is to provide a recombinant expression vector containing a gene sequence encoding the amidase.

[0008] Furthermore, the expression vector can be a commercially available plasmid, granule, phage, etc., constructed by linking the amidase gene sequence into it using molecular biology techniques, with pET30a being the preferred option.

[0009] A third object of the present invention is to provide recombinant cells containing a gene sequence encoding the amidase.

[0010] Furthermore, the recombinant cells are non-plant cells, preferably microorganisms, more preferably any one of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, Lactococcus lactis, Corynebacterium glutamicum, or yeast, with Escherichia coli BL21DE3 being the most preferred.

[0011] A fourth object of the present invention is to provide a recombinant Escherichia coli in which the amidase is overexpressed.

[0012] Furthermore, the recombinant E. coli overexpresses a molecular chaperone; the molecular chaperone includes GroEL and GroES, and the plasmid expressing the molecular chaperone includes pGro7.

[0013] The present invention also provides a method for constructing the recombinant Escherichia coli, comprising the following steps:

[0014] (1) The fully synthesized nucleotide sequence of the amidase gene Afuh is shown in SEQ ID NO.2;

[0015] (2) The amidase gene Afuh was ligated into the vector pET30a to obtain the recombinant plasmid pET30a-Afuh;

[0016] (3) The recombinant plasmid obtained in step (2) was introduced into Escherichia coli BL21DE3 to obtain recombinant Escherichia coli BL21DE3 (pET30a-Afuh). The expression of the Afuh gene in the recombinant bacteria was regulated by the T7 promoter.

[0017] (4) The molecular chaperone plasmid pGro7 was introduced into recombinant Escherichia coli BL21DE3(pET30a-Afuh) to obtain recombinant Escherichia coli BL21DE3(pET30a-Afuh-pGro7) containing the molecular chaperone.

[0018] (5) A recombinant amidase with multiple tolerances was produced by fermentation of recombinant strain BL21DE3 (pET30a-Afuh-pGro7) to degrade ethyl carbamate and acrylamide.

[0019] A fifth objective of the present invention is to provide a method for preparing the amidase, comprising the following steps: obtaining it by fermentation using the recombinant Escherichia coli.

[0020] Furthermore, the recombinant Escherichia coli fermentation includes: culturing the recombinant Escherichia coli to a certain cell concentration (e.g., OD). 600 =0.6-0.8), add inducers (such as IPTG final concentration of 0.5mM and L-arabinose final concentration of 4mg / mL), and culture for a certain time (such as 10-30h) to efficiently express carbamate hydrolase with multiple tolerance.

[0021] A sixth object of the present invention is to provide a composition (such as an enzyme composition) containing the amidase. It may also contain other enzymes with degradative activity.

[0022] A seventh object of the present invention is to provide the use of the amidase, recombinant expression vector, recombinant cell, recombinant Escherichia coli or composition in the degradation of amide compounds or carbamate compounds.

[0023] Furthermore, the amide compounds include, but are not limited to, benzamide, acetamide, acrylamide, butyramide, L-asparagine, urea, etc.

[0024] Furthermore, the carbamate compounds include, but are not limited to, methyl carbamate, ethyl carbamate, tert-butyl carbamate, etc.

[0025] An eighth object of the present invention is to provide the use of the said amidase, recombinant expression vector, recombinant cell, recombinant Escherichia coli, or composition in the preparation of fermentation products. Specifically, it is used for the degradation of amide compounds or carbamate compounds in fermentation products.

[0026] Furthermore, it is used in fermented foods and alcoholic beverages, such as degrading carbamate compounds in heat-processed foods (e.g., French fries, bread, biscuits, instant coffee), alcoholic beverages, etc.

[0027] The beneficial effects of this invention are:

[0028] The amidase obtained in this invention exhibits highly efficient degradation of ethyl carbamate and the carcinogen acrylamide. This enzyme is currently the highest-activity and most stable EC hydrolase reported under acidic conditions (pH 4-5); it is also currently the highest-activity EC hydrolase reported under high-salt conditions (12-18%, v / w); and it exhibits high activity and stability under 10% v / v ethanol conditions. These superior properties facilitate its application in the degradation of ethyl carbamate or acrylamide in high-salt traditional fermented foods and low-alcohol beverages. The enzyme was expressed in *E. coli*. After fermentation and cell disruption, the recombinant bacteria achieved an enzyme activity of 5 U / L with ethyl carbamate as a substrate, and 175 U / L after co-expression with a molecular chaperone. After purification by Ni-NTA affinity chromatography, the specific enzyme activity was 397.5 U / mg with ethyl carbamate as a substrate and 4034.6 U / mg with acrylamide as a substrate at pH 7.0. This enzyme exhibits good acid tolerance, retaining 40%-86% of its activity after being placed at 4°C for 6 hours at pH 4-5. It also demonstrates extremely high salt tolerance, maintaining 100% activity in 10% (v / w) NaCl and 75% activity in 18% (v / w) NaCl. Furthermore, it exhibits good ethanol tolerance, retaining approximately 70% activity in 10% (v / v) ethanol. The recombinant enzyme prepared in this invention lays the foundation for the reduction of EC and acrylamide in high-salt traditional fermented foods and low-alcohol beverages, and for the industrial production of EC hydrolases, yielding significant economic and social benefits. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the construction of the expression plasmid pET30a-Afuh of this invention;

[0030] Figure 2The images show SDS-PAGE images of intracellular proteins in the recombinant bacteria of this invention after fermentation. (A) SDS-PAGE electrophoresis image of E. coli BL21(DE3) / pET30a-Afuh; (B) SDS-PAGE image of intracellular proteins in the recombinant bacteria after co-expression of molecular chaperones; (C) Image showing the expression levels of intracellular proteins in the recombinant bacteria after co-expression of molecular chaperones.

[0031] Figure 4 The diagram shows the composite structure of the amidase AfUH, EC, and acrylamide after molecular docking; the AfUH structure is obtained through homology modeling. (A) Composite structure of AfUH and EC after molecular docking; (B) Composite structure of AfUH and acrylamide after molecular docking.

[0032] Figure 3 This demonstrates the substrate specificity of the recombinant amidase of the present invention.

[0033] Figure 5 Salt tolerance of the recombinant amidase AfUH of the present invention.

[0034] Figure 6 Ethanol tolerance of the recombinant amidase AfUH of the present invention. (A) Relative enzyme activity of AfUH under different ethanol concentrations, (B) Stability of AfUH at different ethanol concentrations;

[0035] Figure 7 The optimal pH and pH stability of the recombinant amidase AfUH of this invention are shown in Figures A, B, and C.

[0036] Figure 8 The optimal temperature and temperature stability of the recombinant amidase AfUH of this invention are shown in Figures A and B. (A) Optimal temperature, (B) Temperature stability. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] The solution involved in this invention is as follows:

[0039] This invention provides an amidase that exhibits stronger substrate degradation activity than previously reported amidases, including amides, carbamates, urea, etc. In some embodiments, the amidase of this invention exhibits stronger substrate specificity, particularly for amides, than previously reported amidases. In some embodiments, the amidase of this invention exhibits stronger salt tolerance than previously reported amidases. In some embodiments, the amidase of this invention exhibits stronger acid tolerance than previously reported amidases. In some embodiments, the amidase of this invention exhibits stronger thermal stability than previously reported amidases.

[0040] Specifically, this invention yields a novel amidase that exhibits high activity and stability under acidic conditions (pH 4-5) (retaining 18%, 40%, and 67% enzyme activity at pH 4.0, 4.5, and 5.0, respectively, and maintaining 40%, 79%, and 86% enzyme activity after 6 hours at 4°C), making it the EC hydrolase with the highest activity and stability under acidic conditions discovered to date. This enzyme also exhibits extremely high catalytic activity in high-salt (NaCl, 18% w / v) environments (maintaining 75% enzyme activity), making it the EC hydrolase with the highest salt tolerance reported to date. Furthermore, this enzyme maintains high activity and stability in a 10% (v / v) ethanol system (retaining 70% enzyme activity in a 10% ethanol solution). Finally, this enzyme also degrades acrylamide. Therefore, this enzyme has great application value in the degradation of EC in traditional high-salt fermented foods (such as soy sauce) and low-alcohol beverages (such as rice wine), as well as in the degradation of acrylamide in heat-processed foods such as fried, baked, and instant coffee.

[0041] Of course, those skilled in the art will recognize that recombinant enzymes that undergo at least one substitution or a set of substitutions also possess the above characteristics. Specifically, the amino acid sequence of the recombinant enzyme is obtained by substituting, deleting, or adding one or more amino acids based on SEQ ID NO.1, and encodes a protein with ethyl carbamate degradation activity. For example, adding or deleting one or more amino acid residues at the C-terminus or N-terminus, or adding a fusion tag, although modifying the form, does not change the enzyme activity of the protein.

[0042] The present invention also provides a polynucleotide sequence encoding the amidase provided herein. In some embodiments, the polynucleotide encodes the amidase provided herein. In some other embodiments, the polynucleotide sequence is codon-optimized.

[0043] This invention provides at least one recombinant protein. In some embodiments, the amidase carries any protein tag. In some embodiments, the N-terminus of the amidase is fused with a protein tag. In other embodiments, the addition of a protein tag can increase the expression level and activity of the protein.

[0044] The present invention also provides expression vectors comprising the polynucleotide sequences provided herein. In some embodiments, the expression vector carries the polynucleotide sequences provided herein. In other embodiments, the expression vector further comprises at least one control sequence. In other embodiments, the control sequence comprises a promoter. In some embodiments, the promoter is a heterologous promoter.

[0045] The present invention also provides host cells transformed with at least one polynucleotide sequence provided herein. In some embodiments, the host cells are transformed with a polynucleotide sequence provided herein.

[0046] The present invention also provides a composition, in some embodiments of which is an enzyme composition containing other enzymes having degradation activity of amide compounds or carbamate compounds.

[0047] The present invention also provides for use with any of the compositions provided herein. In some embodiments, any composition is provided, alone or in any combination. The invention is not intended to be limited to any particular use.

[0048] The sequence information involved in this invention is as follows:

[0049] The amino acid sequence of amidase AfUH (SEQ ID NO.1):

[0050] MKIHRPTREQFVAMYTKMGLNFTENEELEYLIELDKLICHYDKLNIFKDNIPRVKYQRSPGFHPEEVDNKYNAWYVKTNIVGSSSGVLLGKKIAIKDNVAISGVNMMNGASTLEGFIPDIDATIVERILDAGGTILGKSSCEYFCMSGGSHTGALGPVINPYKEGYSAGGSSSGSGVLVATKEVDMAIGGDQGGSIRIPASFCGIYGMKPTWGLVPYTGVMPIENTLDHVGPMTNTVEDNAILLEVLAGSDGLDPRQPSPCSYQSKYGEYTKNLESGVAGLKIAVIDEGFGWECSENDVDDAVKKAANIFVEEGAIVDMVSVPWHRDGISVWTSIASEGSQTQMMNGNAMGFGWKGYYSVNLLDAHAGWRNRADELSESLKLTMLLGQYFIDHYNGRFYAKSQNLARSLRRAYDDVLLDYDILLMPTTPMKAQPLPDISEQLHISVKRAFEPLINTAPFDVTGHPAMSVPCGSSDGLPIGMMLVAKHFDEATIYRAAYAYERASLL

[0051] Nucleotide sequence of amidase AfUH (SEQ ID NO.2):

[0052]

[0053] The materials and methods used in the following embodiments:

[0054] (1) LB medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L (solid medium with 1.5% agar powder added).

[0055] (2) The molecular chaperone plasmid pGro7 (containing the molecular chaperone GroEL and GroES encoding genes) is the commercial plasmid Takara Code No. 3340.

[0056] (3) The following commercially available plasmids and E. coli are used for gene cloning and expression:

[0057] pET30a (Novagen, USA)

[0058] E. coli BL21(DE3) (Novagen, USA)

[0059] (4) Method for determining the activity of ethyl carbamate hydrolase: Take two 1.5 mL EP tubes and add 200 μL of enzyme solution and 200 μL of inactivated enzyme solution to each tube. Then, add 200 μL of 3% EC substrate solution (prepared with 20 mM pH 7.0 phosphate buffer) to each of the two tubes. After reacting in a 37°C water bath for 15 min, add 200 μL of stop agent (10% trichloroacetic acid) to each tube, mix well, and then add 200 μL of colorimetric reagent I (15 g phenol and 0.625 g sodium nitrosoferricyanide diluted to 250 mL with ultrapure water) and 200 μL of colorimetric reagent II (13.125 g NaOH and 7.5 mL NaClO diluted to 250 mL with ultrapure water). Shake vigorously and continue to incubate in a 37°C water bath for 15 min. Remove the tubes and measure the OD value at 625 nm to calculate the enzyme activity (using ammonium chloride to plot a standard curve).

[0060] (5) Definition of ethyl carbamate hydrolase activity unit: Under normal pressure, 20mM citrate-disodium hydrogen phosphate buffer at pH 4.5 and 37°C, the enzyme activity unit is the amount of ethyl carbamate hydrolyzed to produce 1 μmol NH4 per minute. + The required amount of enzyme is one unit of enzyme activity.

[0061] Example 1: Construction of recombinant expression strain of amidase

[0062] The process of constructing recombinant expression vectors is as follows: Figure 1As shown in the figure, the amidase gene sequence was artificially synthesized at Shanghai Sangon Biotech Co., Ltd. The specific process is as follows: Based on the amino acid sequence of the amidase from *Acidithiobacillus ferrianus* (GeneBank Accession number: WP_163098063.1, SEQ ID NO.1) in NCBI, the sequence of the amidase encoding gene (named Afuh in this invention) was optimized using the *E. coli* expression system. The optimized gene sequence is shown in SEQ ID NO.2. The amidase AfUH in this invention has an amino acid sequence similarity of no more than 45% with other currently reported enzymes with EC degradation activity, thus possessing novelty.

[0063] The synthesized gene was digested with Nde I and Xho I, and after gel extraction and recovery, ligated to the pET30a plasmid digested with Nde I and Xho I. The ligation was then performed into E. coli BL21DE3 competent cells, plated on LB agar plates containing 50 μg / ml kanamycin, and incubated at 37°C for 12 h. Positive clones were identified by PCR using forward primer F and reverse primer R. (Amplification system: 20 μl reaction volume was prepared according to the 2×tag kit instructions. Amplification conditions were: 95°C for 3 min per cycle, 95°C for 30 s, 55°C for 30 s, 72°C for 2 min for 30 cycles, and 72°C for 10 min per cycle.) Positive clones correctly identified by colony PCR were inoculated into LB liquid medium containing 50 μg / ml kanamycin, and plasmids were extracted and verified by double enzyme digestion. Plasmids verified by Nde I and Xho I double digestion were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Thus, the exogenous expression system of recombinant ethyl carbamate hydrolase BL21(DE3) / pET30a-afuh was obtained.

[0064] Forward primer F: 5'-CCATGGCAAAAAGCCCGACCAAAGC-3'

[0065] Reverse primer R: 5'-CAGAACCGTCATCACCACCACCACCACTAAGGATCC-3'

[0066] Example 2: Recombinant expression of ethyl carbamate hydrolase

[0067] Single colonies of the genetically engineered bacterium BL21(DE3) / pET30a-Afuh were picked and inoculated into 25 mL of LB broth containing 50 μg / mL kanamycin, and incubated overnight at 37°C with shaking. The next day, the colonies were transferred at a 2% inoculum to TB broth containing 50 μg / mL kanamycin and cultured until the bacterial concentration reached OD500. 600When the concentration of the target cell line was 0.6, IPTG was added to a final concentration of 0.5 mmol / L for induction. The cells were cultured at 20°C for 24 h, collected by centrifugation, and resuspended in 20 mM pH 7.0 phosphate buffer. The cells were then disrupted by sonication, centrifuged again, and the supernatant was collected. The protein content and EC hydrolase activity in the supernatant were measured, and protein expression was detected by SDS-PAGE. Figure 2 A). The results showed that the recombinant cells exhibited weak enzyme activity (5 U / L) on EC after lysis. SDS-PAGE analysis revealed that the recombinant protein was almost entirely present in inclusion body form, resulting in extremely low activity.

[0068] Example 3: Expression and purification of soluble activity of ethyl carbamate hydrolase

[0069] Molecular chaperone plasmids pTf16, pKJE7, and pGro7 were transformed into *E. coli* BL21(DE3) / pET30a-Afuh to obtain recombinant bacteria BL21(DE3) / pET30a-Afuh-pTf16, BL21(DE3) / pET30a-Afuh-pKJE7, and BL21(DE3) / pET30a-Afuh-pGro7, respectively. The recombinant bacteria were inoculated into 25 mL of LB broth containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured overnight at 37°C with shaking. The next day, they were transferred at a 2% inoculum to TB broth containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured until the bacterial concentration reached OD500. 600 When the concentration of the culture medium was 0.6, IPTG was added to a final concentration of 0.5 mmol / L for induction. The cells were cultured at 20°C for 24 h, collected by centrifugation, and resuspended in 20 mM pH 7.0 phosphate buffer. After fermentation, the recombinant bacteria were subjected to ultrasonic disruption, centrifugation, and the supernatant was collected. The protein content and EC hydrolase activity in the supernatant were measured, and protein expression was detected by SDS-PAGE. Figure 2 B). The results showed that the soluble expression level of AfUH in the recombinant strain BL21(DE3) / pET30a-Afuh-pGro7 was greatly enhanced. Enzyme activity assays showed that the crude enzyme activity against EC was 175 U / L at this time. Figure 2 C), while other molecular chaperones did not promote the soluble expression of AfUH. Figure 2 A, B).

[0070] After expression on the recombinant plasmid, the recombinase protein contains a 6× his-tag protein at its C-terminus, therefore HisTrap can be used. TMProtein purification was performed using a 5 mL FF Ni-NTA affinity chromatography column. Equilibration buffer A consisted of 20 mM phosphate, 500 mM NaCl, and 20 mM imidazole, pH 7.4; elution buffer B consisted of 20 mM phosphate, 500 mM NaCl, and 500 mM imidazole, pH 7.4. The flow rate was 5 mL / min, and a gradient elution method (8% B, 20% B, 60% B, 100% B) was used to elute the protein. The target protein was eluted using a 60% B gradient. The eluent was desalted by dialyzing and then analyzed by SDS-PAGE and specific enzyme activity assays. Enzyme activity assays showed that the purified enzyme had a specific activity of 397.5 U / mg.

[0071] Example 4: Substrate specificity of recombinant amidases

[0072] The AfUH enzyme solution obtained in Example 3, after co-expression and purification with the molecular chaperone plasmid pGro7, was mixed with methyl carbamate, EC, tert-butyl carbamate, benzamide, acetamide, acrylamide, butyramide, L-asparagine, and urea, respectively. The corresponding enzyme activities were determined according to the enzyme activity assay method described in the Materials and Methods section. The enzyme activity against EC was taken as 100%, and the relative enzyme activities of AfUH against other substrates were calculated. Enzyme activity detection is as follows... Figure 3 As shown, the enzyme's relative activities for carbamates are similar, indicating that its catalytic efficiency for carbamates is similar to that for EC. The enzyme's activity for amides is significantly higher than its activity for carbamate substrates, with relative activities of 690%, 1015%, and 1770% for acetamide, acrylamide, and butyramide, respectively, indicating that enzyme activity gradually increases with the increase of the amide-terminal side chain. It exhibits activity against EC, a Group 2A carcinogen, and also shows high activity against acrylamide, another Group 2A carcinogen. It has a weak hydrolytic activity of 9 U / L against urea.

[0073] Homology modeling and molecular docking with the amidase AfUH were performed using the online websites I-TASSER (https: / / zhanggroup.org / I-TASSER / ) and BSP-SLIM (https: / / zhanggroup.org / BSP-SLIM / ), respectively, resulting in complexes of AfUH with EC and acrylamide molecules. Figure 4 A, B), which shows that both EC and acrylamide molecules can bind well into the substrate pocket of the enzyme and be degraded by the enzyme, which is consistent with the experiment showing that the enzyme can degrade EC and acrylamide.

[0074] Example 5: Salt tolerance of recombinant amidase

[0075] The purified enzyme AfUH obtained in Example 3 was added to phosphate buffer (20 mM, pH 7.0) containing 3% EC and different mass concentrations (0-18%, w / v) of NaCl. The reaction was carried out at 37°C for 15 min, and then a stop agent was added to determine the enzyme activity. The enzyme activity measured when the substrate solution did not contain NaCl was taken as 100% (specific enzyme activity of 397.5 U / mg). The enzyme activity detection results are as follows: Figure 5 As shown. Figure 5 It was found that NaCl actually promoted the activity of this enzyme at salt concentrations (0-5%), with a relative activity of 325% in the presence of 1% NaCl. This enzyme exhibits extremely high salt tolerance, maintaining 100% activity in the presence of 10% NaCl and 75% activity in the presence of 18% NaCl. Compared to the previously discovered amidase ApUH (patent number: CN 202410286207.3), which maintains 65% activity in the presence of 15% NaCl, AfUH demonstrates significantly superior salt tolerance. In conclusion, this enzyme exhibits extremely high salt tolerance, making it the most salt-tolerant EC hydrolase discovered to date, and possesses the potential for application in EC degradation in traditional fermented foods (such as soy sauce).

[0076] Example 6: Optimal pH and pH stability of recombinant amidase

[0077] The enzyme activity of AfUH was measured under the following conditions: pH 3.0–8.0 (20 mM citrate-disodium hydrogen phosphate) and pH 6.5–9.0 (20 mM disodium hydrogen phosphate-potassium dihydrogen phosphate). The highest enzyme activity obtained was taken as 100%, and the enzyme activity measured under other pH conditions was taken as a percentage of the maximum enzyme activity. The enzyme activity measurement results are as follows: Figure 6As shown in Figure A, this enzyme retains 18%, 40%, 67%, 83%, and 98% of its activity at pH 4.0, 4.5, 5.0, 5.5, and 6.0, respectively, with an optimal pH of 6.5. This compares to the previously discovered amidase ApUH (patent number: CN 202410286207.3), which retains 3%, 4%, 10%, 23%, 60%, and 85% of its activity at pH 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5, respectively, with an optimal pH of 7. The specific enzyme activity of AfUH is 397.5 U / mg (37℃, pH 7.0), while the specific enzyme activity of ApUH (patent number: CN 202410286207.3) is 284.1 U / mg (37℃, pH 7.0). Comparative studies revealed that AfUH has a lower optimal pH than ApUH and retains high enzyme activity under acidic conditions. The amidase from *Saccharomyces cerevisiae* showed no activity at pH < 5.0 (Patent No.: ZL202210528649). These results indicate that the amidase obtained in this invention is more advantageous for EC degradation in acidic fermented foods and alcoholic beverages.

[0078] To determine pH stability, the purified amidase was added to buffer solutions of different pH values ​​(pH range 3.0-9.0) with an initial enzyme activity of 100%. After incubation at 4°C for 6 hours, the residual enzyme activity was measured. The enzyme activity results are as follows: Figure 6 As shown in Figure B, this enzyme is relatively stable between pH 5.5 and 8.0, retaining over 80% of its activity after treatment. Furthermore, after 6 hours of incubation at pH 4.0, 4.5, 5.0, and 5.5, it retains approximately 20%, 43%, 70%, and 85% of its activity, respectively, demonstrating a certain degree of acid tolerance. In contrast, the amidase ApUH (patent number: CN 202410286207.3) retains approximately 20%, 42%, and 58% of its activity after 6 hours of incubation at pH 4.0, 4.5, and 5.0, respectively. This comparison shows that AfUH is more stable under acidic conditions.

[0079] The optimal pH and pH stability determination results of AfUH indicate that this enzyme currently exhibits the highest activity and stability of EC hydrolase under acidic conditions (pH 4.0-5.0). This enzyme is suitable for the degradation of EC in traditionally fermented foods in weakly acidic, neutral, and weakly alkaline conditions, and has significant application value in high-salt and low-alcohol beverages.

[0080] Example 7: Ethanol tolerance of recombinant amidase

[0081] The enzyme activity of AfUH was measured under conditions containing 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40% (v / v) ethanol, respectively. The enzyme activity measured under ethanol-free conditions was taken as 100%. The relative enzyme activity of amidase under different ethanol concentrations was determined. The results are as follows: Figure 7 As shown in A, the enzyme still retains 70%, 20%, 10%, and 5% enzyme activity in ethanol containing 10%, 20%, 30%, and 40% (v / v), respectively.

[0082] AfUH was added to phosphate buffer (20 mM, pH 7.0) containing 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40% (v / v) ethanol, respectively. After incubation at 37°C for 1 hour, enzyme activity was measured. Enzyme activity measured using enzyme solutions treated with 0% ethanol was considered 100%. The results are as follows: Figure 7 As shown in B, the enzyme can still retain 80% or more of its activity after being placed at 37°C for 1 hour with less than 5% (v / v) ethanol. It has certain stability in environments containing low concentrations of ethanol and has the potential to be applied to the degradation of EC in alcoholic beverages.

[0083] Example 8: Optimal temperature and temperature stability of recombinant amidase

[0084] The activity of AfUH was measured at 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, and 90 °C. The highest enzyme activity was defined as 100%, and the relative enzyme activities of the other groups were calculated. The temperature corresponding to the highest enzyme activity was taken as the optimum temperature. Figure 8 As shown in Figure A, the optimal temperature is 50℃.

[0085] AfUH enzyme solutions were incubated at 20, 30, 40, 50, 60, 70, 80, and 90°C for 30 minutes, and their residual enzyme activity was immediately measured. Using the enzyme activity of the untreated enzyme as 100%, the relative enzyme activity of each incubated group was calculated. The temperature stability results are shown below. Figure 8 As shown in Figure B, this enzyme exhibits good thermal stability, retaining over 100% of its activity after incubation at 40°C or lower for 30 minutes. However, its thermal stability decreases sharply above 40°C. Given that this enzyme's application environment does not include high-temperature environments, temperature has a relatively small impact on its practical application.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of amidase in the degradation of amide compounds or carbamate compounds; the gene sequence encoding the amidase is shown in SEQ ID NO.2; The amide compounds include benzamide, acetamide, acrylamide, butyramide, L-asparagine, or urea; The carbamate compounds include methyl carbamate, ethyl carbamate, or tert-butyl carbamate.

2. Application of recombinant *Escherichia coli* in the degradation of amide compounds or carbamate compounds; wherein the recombinant *E. coli* overexpresses amidase; wherein the recombinant *E. coli* overexpresses molecular chaperones; wherein the molecular chaperones include GroEL and GroES carried by the pGro7 plasmid; the gene sequence encoding the amidase is shown in SEQ ID NO.2; The amide compounds include benzamide, acetamide, acrylamide, butyramide, L-asparagine, or urea; The carbamate compounds include methyl carbamate, ethyl carbamate, or tert-butyl carbamate.

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