Amidase with multiple tolerance and capable of degrading carbamate and amide compounds

Abstract

The invention discloses an amidase with multiple tolerance and capable of degrading carbamate and amide compounds. The enzyme can efficiently degrade carbamate and amide compounds, and has extremely high salt tolerance and acid tolerance. According to the invention, high-efficiency expression of amidase genes and purification of zymoprotein are successfully realized by utilizing an escherichia coli expression system, and the specific enzyme activity of pure enzyme is up to 397.5 U / mg. When the concentration of NaCl is not higher than 5% (w / v), the salt has a great promotion effect on the activity of the enzyme, and 75% or more of the enzyme activity can still be maintained under the condition that the concentration of NaCl is not higher than 18% (w / v); according to the present invention, the enzyme activity can be maintained at 40% under the condition of pH 4.5, and the enzyme activity can be maintained at about 43-85% after the enzyme is placed for 6 h under the condition of pH 4.0-5.0, such that the strong acid tolerance is provided; meanwhile, certain ethanol tolerance is realized. The amidase prepared by the method lays a foundation for eliminating impurities in high-salt fermented food and low-alcohol alcoholic beverages, and has huge economic and social benefits.

Description

Technical Field

[0001] The present invention relates to an amidase with multiple tolerances and capable of degrading carbamate and amide compounds, belonging to the field of biotechnology. Background Art

[0002] Ethyl carbamate (EC), also known as urethane, is a harmful nitrogen-containing metabolite commonly found in fermented foods and alcoholic beverages. It has genotoxicity and strong carcinogenicity and is widely present in traditional fermented foods and alcoholic beverages such as yellow rice wine, white liquor, wine, sake, and soy sauce. In 1943, Nettleship first confirmed the carcinogenicity of EC. Subsequently, studies have shown that EC can cause diseases such as 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 probably carcinogenic to humans. The formation of EC is related to precursor substances such as urea and ethanol during the fermentation process, and its content is affected by various factors, including fermentation conditions, storage time, and temperature. To protect the health of consumers, many countries (such as the United States, Canada, etc.) have set strict limit standards for the EC content in foods and beverages. Methods for reducing the EC content include process optimization, metabolic engineering modification of fermentation strains, and enzymatic degradation methods. Among them, the enzymatic degradation method directly decomposes EC into non-toxic ethanol, carbon dioxide, and ammonia by using specific enzymes such as EC hydrolase. Without affecting the flavor of traditional fermented foods, the enzymatic degradation method can efficiently degrade ethyl carbamate formed by all pathways. In theory, compared with other methods, this method is the most promising method to completely solve the food safety problem caused by EC in traditional fermented foods in the future.

[0003] However, there is currently no enzyme that can be effectively applied to directly degrade EC in traditional fermented foods (such as soy sauce). The main reason is that the biochemical properties of the currently discovered EC hydrolases have major defects: First, most traditional fermented foods are acidic. However, the currently discovered ethyl carbamate hydrolases have poor acid tolerance, low enzyme activity, and extremely poor stability under the conditions of pH 3-5, severely limiting their application in traditional fermented foods. Second, the currently discovered EC hydrolases have poor salt tolerance and extremely low or even lose their activity in an environment containing a high salt (NaCl, 18% w / v) concentration, thus limiting their application in high-salt fermented foods such as soy sauce. Third, the currently discovered EC hydrolases have poor salt-alcohol tolerance and have low activity and poor stability in the presence of ethanol (10% v / v). Therefore, screening out EC hydrolases with high activity and stability in an acidic environment is the key to solving the problem of EC degradation in traditional fermented foods. At the same time, if the screened enzyme also has high EC degradation activity and stability in a high-salt or ethanol-containing environment, it will strengthen its application in high-salt traditional fermented foods or alcoholic beverages.

[0004] Acrylamide is an organic compound classified as a Group 2A "probable human carcinogen" by the International Agency for Research on Cancer. It mainly exists in thermally processed foods such as frying and baking, such as French fries, bread, biscuits, and instant coffee. It is produced by the Maillard reaction during the high-temperature heating of foods rich in carbohydrates and amino acids, and its formation pathways mainly include the asparagine pathway and the acrolein pathway. Since acrylamide has neurotoxicity, reproductive toxicity, and genotoxicity and may be potentially associated with the occurrence of cancer, it is necessary to reduce its content. Enzymatic direct degradation of acrylamide has the advantages of high efficiency, specificity, and mild conditions, and can reduce the acrylamide content without significantly affecting the food quality, which is an ideal degradation method. Summary of the Invention

[0005] To solve the above problems, the present invention provides an amide with multiple tolerances and capable of degrading ethyl carbamate and acrylamide and its application, to solve the problems of low activity and poor stability of ethyl carbamate hydrolase under acidic, high-salt, and ethanol-containing conditions, and to provide a new method for EC degradation in traditional fermented foods and acrylamide degradation in thermally processed foods such as frying and baking.

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

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

[0008] Furthermore, the expression vector can be constructed by ligating the amidase gene sequence into commercially available plasmids, cosmids, phages, etc. by molecular biology techniques, and pET30a is preferably used.

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

[0010] Furthermore, the recombinant cell is a non-plant cell, preferably a microorganism, more preferably any one of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, Lactococcus lactis, Corynebacterium glutamicum, or yeast, and Escherichia coli BL21DE3 is preferably used.

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

[0012] Furthermore, molecular chaperones are overexpressed in the recombinant Escherichia coli; the molecular chaperones include GroEL and GroES, and the plasmid expressing the molecular chaperones includes pGro7.

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

[0014] (1) Synthesize the amide enzyme gene Afuh with the nucleotide sequence as shown in SEQ ID NO.2 by total gene synthesis;

[0015] (2) Connect the amide enzyme gene Afuh to the vector pET30a to obtain the recombinant plasmid pET30a-Afuh;

[0016] (3) Introduce the recombinant plasmid obtained in step (2) into Escherichia coli BL21DE3 to obtain the recombinant Escherichia coli BL21DE3(pET30a-Afuh), and the expression of the Afuh gene in the recombinant bacteria is regulated by the T7 promoter;

[0017] (4) Introduce the molecular chaperone plasmid pGro7 into the recombinant Escherichia coli BL21DE3(pET30a-Afuh) to obtain the recombinant Escherichia coli BL21DE3(pET30a-Afuh-pGro7) containing the molecular chaperone;

[0018] (5) Use the recombinant bacteria BL21DE3(pET30a-Afuh-pGro7) to ferment and produce a recombinant amide enzyme with multiple tolerances and capable of degrading ethyl carbamate and acrylamide.

[0019] The fifth object of the present invention is to provide a method for preparing the amide enzyme, which includes the following steps: obtained by fermenting the recombinant Escherichia coli.

[0020] Further, the fermentation of the recombinant Escherichia coli includes: culturing the recombinant Escherichia coli to a certain cell concentration (such as OD 600 = 0.6 - 0.8), adding an inducer (such as IPTG with a final concentration of 0.5 mM and L-arabinose with a final concentration of 4 mg / mL), culturing for a certain time (such as 10 - 30 h), and highly expressing an ethyl carbamate hydrolase with multiple tolerances.

[0021] The sixth object of the present invention is to provide a composition (such as an enzyme composition), and the composition contains the amide enzyme. It may also contain other enzymes with degradation activity.

[0022] The seventh object of the present invention is to provide the application of the amide enzyme, recombinant expression vector, recombinant cell, recombinant Escherichia coli or composition in degrading amide compounds or carbamate compounds.

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

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

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

[0026] Further, it is used for fermented foods and alcoholic beverages, such as degrading carbamate compounds in soy sauce, vinegar, pickles, thermally processed foods such as frying and baking (such as French fries, bread, biscuits, instant coffee), wines or alcoholic beverages, etc.

[0027] Advantages of the present invention:

[0028] The amidase obtained in the present invention has the function of efficiently degrading ethyl carbamate and the carcinogen acrylamide; this enzyme is the EC hydrolase with the highest activity and stability reported so far under acidic (pH 4-5) conditions; this enzyme is the EC hydrolase with the highest activity reported so far under high salt (12-18%, v / w) conditions; this enzyme has high activity and stability under 10% v / v ethanol conditions; the above excellent properties contribute to the application of this enzyme in the degradation of ethyl carbamate or acrylamide in high-salt traditional fermented foods and low-alcohol alcoholic beverages. This enzyme is expressed in Escherichia coli. After the recombinant bacteria are fermented and cell lysed, the enzyme activity reaches 5 U / L when using ethyl carbamate as the substrate, and reaches 175 U / L after co-expression with molecular chaperones. After this enzyme is purified by Ni-NTA affinity chromatography, the specific enzyme activity is 397.5 U / mg when using ethyl carbamate as the substrate under pH 7.0 conditions, and the enzyme activity is 4034.6 U / mg when using acrylamide as the substrate. This enzyme has good acid tolerance and can still maintain 40%-86% of the enzyme activity after being placed at 4°C for 6 h under pH 4-5 conditions; this enzyme has extremely high salt tolerance and can maintain 100% of the enzyme activity under the condition of containing 10% (v / w) NaCl, and can still maintain 75% of the enzyme activity under the condition of containing 18% (v / w) NaCl; this enzyme has good ethanol tolerance and still has about 70% of the enzyme activity in the presence of 10% (v / v) ethanol. The recombinant enzyme prepared in the present invention lays a foundation for the reduction of EC and acrylamide in high-salt traditional fermented foods and low-alcohol alcoholic beverages and the industrial production of EC hydrolase, and has great economic and social benefits. Description of the drawings

[0029] Figure 1 It is a schematic diagram of the construction of the expression plasmid pET30a-Afuh of the present invention;

[0030] Figure 2This is the SDS-PAGE diagram of intracellular proteins after fermentation of the recombinant bacteria of the present invention. (A) SDS-PAGE protein electrophoresis diagram of E. coli BL21(DE3) / pET30a-Afuh; (B) SDS-PAGE diagram of intracellular proteins of the recombinant bacteria after co-expression of molecular chaperones; (C) Diagram of the expression level of intracellular proteins of the recombinant bacteria after co-expression of molecular chaperones.

[0031] Figure 4 This is the composite structure diagram of amidase AfUH of the present invention after molecular docking with EC and acrylamide; among them, the structure of AfUH is obtained by homology modeling. (A) Composite structure diagram of AfUH after molecular docking with EC; (B) Composite structure diagram of AfUH after molecular docking with acrylamide;

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

[0033] Figure 5 This is the salt tolerance of the recombinant amidase AfUH of the present invention.

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

[0035] Figure 7 This is the optimal pH and pH stability of the recombinant amidase AfUH of the present invention. (A) Optimal pH, (B) pH stability;

[0036] Figure 8 This is the optimal temperature and temperature stability of the recombinant amidase AfUH of the present invention. (A) Optimal temperature, (B) Temperature stability. Detailed implementation manners

[0037] The following further illustrates the present invention in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not used as a limitation to the present invention.

[0038] The solutions involved in the present invention are as follows:

[0039] The present invention provides an amidase, which has stronger substrate degradation activity than previously reported amidases, and the substrates include amide compounds, carbamate compounds, urea, etc. In some additional embodiments, the amidase of the present invention has stronger substrate specificity than previously reported amidases, especially for amide compounds. In some additional embodiments, the amidase of the present invention has stronger salt tolerance than previously reported amidases. In some additional embodiments, the amidase of the present invention has stronger acid tolerance than previously reported amidases. In some additional embodiments, the amidase of the present invention has stronger thermal stability than previously reported amidases.

[0040] Specifically: A new amidase is obtained in the present invention, which has high activity and stability under acidic (pH 4 - 5) conditions (it still has 18%, 40%, and 67% enzyme activities respectively under the conditions of pH 4.0, 4.5, and 5.0, and can still maintain 40%, 79%, and 86% enzyme activities respectively after being placed at 4°C for 6 hours), and it is the EC hydrolase with the highest activity and stability under acidic conditions currently discovered; this enzyme has extremely high catalytic activity in a high-salt (NaCl, 18% w / v) environment (it can maintain 75% enzyme activity), and it is the EC hydrolase with the highest salt tolerance currently reported; this enzyme still has high activity and stability in a 10% (v / v) ethanol system (this enzyme has 70% enzyme activity in ethanol containing 10%); this enzyme can also degrade acrylamide. Therefore, this enzyme has great application value in the degradation of EC in high-salt traditional fermented foods (such as soy sauce, etc.) and low-alcohol beverages (such as yellow rice wine, etc.), and in the degradation of acrylamide in heat-processed foods such as frying, baking, and instant coffee.

[0041] Of course, those skilled in the art can know that the recombinant enzyme with at least one substitution or substitution set for this enzyme also has the above characteristics. Specifically, the amino acid sequence of the recombinant enzyme is obtained by substituting, deleting, or adding one or several amino acids based on SEQ ID NO.1, and encodes a protein with the function of degrading ethyl carbamate. For example, adding or deleting one or several amino acid residues at the C-terminus or N-terminus, adding a fusion tag, etc., although modified in 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 additional embodiments, the polynucleotide sequence is codon-optimized.

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

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

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

[0046] The present invention also provides a composition which, in some embodiments, is an enzyme composition and contains other enzymes having activity for degrading amide compounds or carbamate compounds.

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

[0048] The sequence information related to the present 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] Materials and methods used in the following examples:

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

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

[0056] (3) The following commercial plasmids and Escherichia 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 enzyme activity of ethyl carbamate hydrolase: Take two 1.5 mL EP tubes, add 200 μL of enzyme solution and 200 μL of inactivated enzyme solution respectively. Then add 200 μL of 3% EC substrate solution (prepared with 20 mM phosphate buffer at pH 7.0) to both tubes. After reacting in a 37°C constant temperature water bath for 15 min, add 200 μL of terminator (10% trichloroacetic acid) to each tube. After mixing, add 200 μL of chromogenic reagent I (15 g phenol and 0.625 g sodium nitroprusside made up to 250 mL with ultrapure water) and 200 μL of chromogenic reagent II (13.125 g NaOH and 7.5 mL NaClO made up to 250 mL with ultrapure water), shake strongly, continue to incubate in a 37°C constant temperature water bath for 15 min and then take out. Measure the OD value at 625 nm and calculate the enzyme activity (using ammonium chloride to draw the standard curve).

[0060] (5) Definition of the enzyme activity unit of ethyl carbamate hydrolase: Under normal pressure, in 20 mM citrate - 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 NH4 + per minute is one enzyme activity unit.

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

[0062] The process of constructing the recombinant expression vector is as Figure 1As shown in the figure. The amidase gene sequence was artificially synthesized by Shanghai Sangon Biotech Co., Ltd. The specific process is as follows: Based on the amidase amino acid sequence (GeneBank Accession number: WP_163098063.1, SEQ ID NO.1) from Acidithiobacillus ferrianus in NCBI, the sequence of the amidase-encoding gene (named Afuh in this invention) was optimized based on the Escherichia coli expression system. The optimized gene sequence is shown in SEQ ID NO.2. The amidase AfUH in this invention has a similarity of no more than 45% with the amino acid sequences of other enzymes that have been reported to have EC degradation effects, and has novelty.

[0063] The synthetic gene was digested with Nde I and Xho I, and after gel extraction and recovery, it was ligated to the pET30a plasmid digested with Nde I and Xho I, and then transferred into E. coli BL21DE3 competent cells. The cells were spread on an LB solid plate containing 50 μg / ml kanamycin and cultured at 37°C for 12 h. Positive clones were identified by PCR using the forward primer F and the reverse primer R. (Amplification system: Prepare a 20 μl reaction system according to the 2×tag kit instructions. Amplification conditions: 95°C for 3 min for 1 cycle, 95°C for 30 s, 55°C for 30 s, 72°C for 2 min, 30 cycles, and 72°C for 10 min for 1 cycle.) The positive clones with correct colony PCR identification were inoculated into an LB liquid medium containing 50 μg / ml kanamycin for culture, and the plasmid was extracted for plasmid double digestion verification. The plasmid verified correctly by double digestion with Nde I and Xho I was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Thus, the heterologous expression system BL21(DE3) / pET30a-afuh of the recombinant ethyl carbamate hydrolase 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] Pick a single colony of the genetic engineering bacterium BL21(DE3) / pET30a-Afuh and inoculate it into 25 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture it overnight at 37°C with shaking. The next day, transfer it to TB medium containing 50 μg / mL kanamycin at an inoculation amount of 2%, and culture it until the cell density OD 600When = 0.6, IPTG was added to a final concentration of 0.5 mmol / L for induction, and the cells were cultured at 20 °C for 24 h. The cells were collected by centrifugation, resuspended with 20 mM phosphate buffer at pH 7.0, broken by ultrasonic treatment, and the supernatant was obtained by centrifugation. The protein content and EC hydrolase activity in the supernatant were measured, and the protein expression was detected by SDS-PAGE( Figure 2 A). The measurement results showed that the recombinant cells had weak enzyme activity (5 U / L) against EC after being broken. SDS-PAGE analysis found that the recombinant protein existed almost entirely in the form of inclusion bodies, resulting in extremely low activity.

[0068] Example 3: Soluble activity expression and purification of ethyl carbamate hydrolase

[0069] The molecular chaperone plasmids pTf16, pKJE7, and pGro7 were respectively transformed into Escherichia coli BL21(DE3) / pET30a-Afuh to obtain recombinant strains BL21(DE3) / pET30a-Afuh-pTf16, BL21(DE3) / pET30a-Afuh-pKJE7, and BL21(DE3) / pET30a-Afuh-pGro7. The recombinant strains were respectively inoculated into 25 mL of LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured overnight at 37 °C with shaking. The next day, they were transferred to TB medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol at an inoculation amount of 2%, and cultured until the cell density OD 600 When = 0.6, IPTG was added to a final concentration of 0.5 mmol / L for induction, and the cells were cultured at 20 °C for 24 h. The cells were collected by centrifugation, resuspended with 20 mM phosphate buffer at pH 7.0. After fermentation, the recombinant bacteria were broken by ultrasonic treatment, and the supernatant was obtained by centrifugation. The protein content and EC hydrolase activity in the supernatant were measured, and the protein expression was detected by SDS-PAGE( Figure 2 B). The measurement results showed that the soluble expression level of AfUH in the recombinant strain BL21(DE3) / pET30a-Afuh-pGro7 was greatly improved. The enzyme activity measurement results showed that the crude enzyme activity of this enzyme against EC was 175 U / L at this time( Figure 2 C), while other molecular chaperones had no promoting effect on the soluble expression of AfUH( Figure 2 A, B).

[0070] After expression by the recombinant plasmid, the recombinant enzyme protein contains a 6×his-tag protein at the C-terminus. Therefore, HisTrap can be used TMProtein purification was carried out using a 5 ml FF Ni-NTA affinity chromatography column. Equilibration buffer A: 20 mM phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4; elution buffer B: 20 mM phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4. The flow rate was 5 mL / min, and gradient elution (8% B, 20% B, 60% B, 100% B) was used to elute the protein. The target protein was eluted at a 60% B gradient. The eluate was desalted by dialysis and then subjected to SDS-PAGE detection and specific enzyme activity detection. By measuring the enzyme activity, it was found that the specific enzyme activity of the purified enzyme reached 397.5 U / mg.

[0071] Example 4: Substrate specificity of recombinant amidase

[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. According to the enzyme activity measurement method described in Materials and Methods, the corresponding enzyme activities were measured. Taking the enzyme activity of the enzyme against EC as 100%, the relative enzyme activities of AfUH against other substrates were converted. The enzyme activity detection was as Figure 3 shown. The relative enzyme activities of this enzyme for carbamate compounds were similar, indicating that the catalytic efficiency of this enzyme for carbamate compounds was similar to that for EC; the enzyme activity of this enzyme for amide compounds was much higher than that for carbamate substrates. The relative activities for acetamide, acrylamide, and butyramide were 690%, 1015%, and 1770% respectively, indicating that as the side chain of the amide end increased, the enzyme activity gradually increased. It was active against the 2A class carcinogen EC and also had relatively high activity against the 2A class carcinogen acrylamide. It had weak hydrolytic activity against urea, 9 U / L.

[0073] Homology modeling of the amidase AfUH and molecular docking with the EC molecule were carried out through the online websites I-TASSER (https: / / zhanggroup.org / I-TASSER / ) and BSP-SLIM (https: / / zhanggroup.org / BSP-SLIM) respectively, and the complexes of AfUH with EC and acrylamide molecules were obtained ( Figure 4 A,B). This indicated that both the EC and acrylamide molecules could bind well to the substrate pocket of this enzyme and thus be degraded by this enzyme, which was also consistent with the fact that this enzyme could degrade EC and acrylamide in the experiment.

[0074] Example 5: Salt tolerance of recombinant amidase

[0075] The pure enzyme AfUH obtained in Example 3 was added to a phosphate buffer (20 mM, pH 7.0) containing 3% EC and different mass concentrations (0 - 18%, w / v) of NaCl. After reacting at 37 °C for 15 min, a terminator was added to measure the enzyme activity. Taking the enzyme activity measured when there was no NaCl in the substrate solution as 100% (specific enzyme activity was 397.5 U / mg), the enzyme activity detection results are as Figure 5 shown. As can be seen from Figure 5 , when the salt concentration was (0 - 5%), NaCl had a promoting effect on the activity of this enzyme. Under the condition of containing 1% NaCl, the relative activity of this enzyme was 325%; this enzyme had extremely high salt tolerance. Under the condition of containing 10% NaCl, it could maintain 100% of the enzyme activity. Under the condition of containing 18% NaCl, it could still maintain 75% of the enzyme activity. Compared with the previously discovered amidase ApUH (patent number: CN 202410286207.3) which had 65% of the enzyme activity under the condition of containing 15% NaCl, it was found that the salt tolerance of AfUH was much better than that of ApUH. In summary, this enzyme had extremely high salt tolerance and was the EC hydrolase with the highest salt tolerance found so far, having the potential to be applied to the degradation of EC in traditional fermented foods (such as soy sauce).

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

[0077] The enzyme activities of AfUH were measured under the conditions of pH 3.0 - 8.0 (20 mM citric acid - disodium hydrogen phosphate) and 6.5 - 9.0 (20 mM disodium hydrogen phosphate - potassium dihydrogen phosphate) respectively. Taking the highest enzyme activity obtained by measurement as 100%, the enzyme activities measured under other pH conditions were the percentages of the maximum enzyme activity. The enzyme activity measurement results are as Figure 6As shown in Figure A, the enzyme still has 18%, 40%, 67%, 83%, and 98% of its enzyme activity at pH values of 4.0, 4.5, 5.0, 5.5, and 6.0, respectively, and the optimal pH of this enzyme is 6.5. The previously discovered amidase ApUH (Patent No.: CN 202410286207.3) still has 3%, 4%, 10%, 23%, 60%, and 85% of its enzyme activity at pH values of 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5, respectively, and the optimal pH is 7. The specific enzyme activity of AfUH is 397.5 U / mg (37°C, pH 7.0), while that of ApUH (Patent No.: CN 202410286207.3) is 284.1 U / mg (37°C, pH 7.0). By comparison, it is found that compared with ApUH, AfUH has a lower optimal pH and retains higher enzyme activity under acidic conditions. The amidase from Lodderomyces elongisporus is inactive under pH < 5.0 conditions (Patent No.: ZL202210528649.). The above results indicate that the amidase obtained in the present invention has more advantages in the degradation of EC in acidic fermented foods and alcoholic beverages.

[0078] When measuring the pH stability, with the initial enzyme activity as 100%, the purified amidase was added to buffer solutions with different pH values (pH measurement range: 3.0 - 9.0), and the residual enzyme activity was measured after standing at 4°C for 6 h. The results of enzyme activity measurement are as Figure 6 shown in Figure B. The enzyme is relatively stable between pH 5.5 - 8.0 and can still maintain more than 80% of its enzyme activity after treatment; at the same time, when the enzyme is placed at pH 4.0, 4.5, 5.0, and 5.5 for 6 h, it can maintain about 20%, 43%, 70%, and 85% of its enzyme activity respectively, showing certain acid tolerance. Amidase ApUH (Patent No.: CN 202410286207.3) can maintain about 20%, 42%, and 58% of its enzyme activity after being placed at pH 4.0, 4.5, and 5.0 for 6 h. By comparison, it can be seen that AfUH has better enzyme stability under acidic conditions.

[0079] The measurement results of the optimal pH and pH stability of AfUH indicate that this enzyme is the EC hydrolase with the highest activity and stability discovered so far under acidic (pH 4.0 - 5.0) conditions. This enzyme is suitable for the degradation of EC in weakly acidic, neutral, and weakly alkaline traditional fermented foods, and has great application value for high-salt and low-alcohol alcoholic beverages.

[0080] Example 7: Ethanol Tolerance of Recombinant Amidase

[0081] The enzyme activity of AfUH was measured under the conditions of containing 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35% and 40% (v / v) ethanol respectively. Taking the enzyme activity measured under the condition without ethanol as 100%, the relative enzyme activity of amidase under different ethanol concentration conditions was measured. The measurement results are as Figure 7 shown in A. The enzyme still had 70%, 20%, 10% and 5% of the enzyme activity respectively in the ethanol containing 10%, 20%, 30% and 40% (v / v).

[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 being placed at 37 °C for 1 h, the enzyme activity was detected. Taking the enzyme activity detected by the enzyme solution treated under the condition of containing 0% ethanol as 100%. The measurement results are as Figure 7 shown in B. After being placed at 37 °C for 1 h with less than 5% (v / v) ethanol, the enzyme could still maintain more than 80% of the enzyme activity. It had certain stability in the environment containing low ethanol concentration and had the potential to be applied to the degradation of EC in alcoholic beverages.

[0083] Example 8: Optimum 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, 90 °C respectively. Taking the enzyme activity of the highest group as 100%, the relative enzyme activity of other groups was calculated. The temperature corresponding to the group with the highest enzyme activity was the optimum temperature. As Figure 8 shown in A, the optimum temperature was 50 °C.

[0085] The AfUH enzyme solution was placed at 20, 30, 40, 50, 60, 70, 80, 90 °C respectively. After being incubated for 30 min, the residual enzyme activity was immediately detected. Taking the enzyme activity of the enzyme without incubation treatment as 100%, the relative enzyme activity of each group of enzymes after incubation treatment was calculated. The temperature stability results are as Figure 8 shown in B. The enzyme had good thermal stability. It could maintain more than 100% of its enzyme activity after being incubated for 30 min at less than or equal to 40 °C. When the temperature was higher than 40 °C, the thermal stability of the enzyme decreased sharply. In view of the application environment of the enzyme did not include high temperature environment, the influence of temperature on its actual application was small.

[0086] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. An amidase, characterized in that The gene sequence encoding the amidase is shown in SEQ ID NO.

2.

2. A recombinant expression vector carrying the gene sequence encoding the amidase according to claim 1.

3. A recombinant cell containing the amidase according to claim 1.

4. A recombinant Escherichia coli, characterized in that The amidase according to claim 1 is overexpressed in the recombinant Escherichia coli.

5. The recombinant Escherichia coli according to claim 4, characterized in that The molecular chaperones are overexpressed in the recombinant Escherichia coli; the molecular chaperones include GroEL and GroES, and the plasmids expressing the molecular chaperones include pGro7.

6. A method for preparing amidase, characterized in that: The method comprises the following steps: obtaining the product by fermentation using the recombinant Escherichia coli described in claim 4 or 5.

7. A composition, characterized in that The composition contains the amidase according to claim 1.

8. Use of the amidase according to claim 1, the recombinant expression vector according to claim 2, the recombinant cell according to claim 3, the recombinant Escherichia coli according to claim 4 or 5, or the composition according to claim 7 in degrading amide compounds or carbamate compounds.

9. The use according to claim 8, characterized in that: Contains at least one of the following characteristics: (1) The amide compound includes benzamide, acetamide, acrylamide, butyramide, L-asparagine or urea; (2) The carbamate compound includes methyl carbamate, ethyl carbamate or tert-butyl carbamate.

10. Use of the amidase according to claim 1, the recombinant expression vector according to claim 2, the recombinant cell according to claim 3, the recombinant Escherichia coli according to claim 4 or 5, or the composition according to claim 7 in the preparation of a fermentation product.

Images