High-salt-tolerant amidase, gene, vector, recombinant bacteria and application thereof

Abstract

The present application relates to the technical field of recombinant protein technology, and particularly relates to a high-salt-tolerant amidase, gene, vector, recombinant bacteria and application, wherein the amino acid sequence of the amidase is shown as SEQ ID NO. 1. The amidase has extremely high salt tolerance and good ethanol tolerance. The recombinant amidase prepared by the present application lays a foundation for future realization of elimination of ethyl carbamate in high-salt fermented food and alcoholic beverages and realization of industrial application of ethyl carbamate hydrolytic enzyme, and has great economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein technology, and more particularly to a high-salt-resistant amidase, gene, vector, recombinant bacteria, and their applications. Background Technology

[0002] Ethyl carbamate (or urethane, abbreviated EC) is a substance with genotoxicity and strong carcinogenicity (it can cause lung cancer, lymphoma, liver cancer, skin cancer, etc.). It is widely found in many fermented foods (such as soy sauce, vinegar, and pickles) and alcoholic beverages (such as rice wine, baijiu, wine, Japanese sake, and brandy). Humans primarily ingest ethyl carbamate through alcoholic beverages and food. Ethyl carbamate has become a significant factor affecting human health. Therefore, it is urgent to adopt effective methods to eliminate ethyl carbamate from fermented foods and alcoholic beverages.

[0003] Ethyl carbamate hydrolases can directly and efficiently degrade ethyl carbamate (EC) into non-toxic ethanol, ammonia, and carbon dioxide, making them the most promising method to completely solve the food safety problems caused by EC in traditional fermented foods. However, currently, there are no effective enzymes for the direct degradation of EC in traditional fermented foods (such as soy sauce), mainly due to significant defects in the biochemical characteristics of the EC hydrolases discovered so far: First, because urea and EC have similar molecular structures, most of the currently discovered amidase-type EC hydrolases can simultaneously degrade urea and EC. The urea content (approximately 50 mg / L) in fermented foods is 50-1000 times higher than the EC content (10-750 μg / L), and the huge substrate competition between them results in poor performance of these EC hydrolases when applied to the direct degradation of EC. Second, the currently discovered EC hydrolases have poor salt tolerance, exhibiting extremely low activity or even loss of activity in environments containing high salt concentrations (NaCl, 10-20%, w / v), thus limiting their application in high-salt fermented foods such as soy sauce. Therefore, screening out EC hydrolases that are non-degradable urea and highly active in high-salt environments is key to solving the bottleneck problem of EC reduction in traditional high-salt fermented foods. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a high-salt-resistant amidase, gene, vector, recombinant bacteria, and application to solve the problem.

[0005] To achieve the above objectives, the present invention provides a high-salt-tolerant amidase, the amino acid sequence of which is shown in SEQ ID NO.1. This amidase exhibits good salt tolerance in high-salt (NaCl, 10-20%, w / v) environments and ethanol tolerance.

[0006] The amino acid sequence of the high-hydrochloride-resistant amidase is obtained by substituting, deleting, or adding one or more amino acids, and it encodes a protein with urethane 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, may modify the form but does not change the protein's enzymatic activity.

[0007] The present invention also provides a gene encoding the high-salt-tolerant amidase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant expression vector containing the said gene.

[0009] In one embodiment of the present invention, 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 pRSFDuet-1 being preferred.

[0010] The present invention also provides a recombinant bacterium containing the recombinant expression vector.

[0011] Optionally, the host bacteria expressing the gene can be any one of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, Lactococcus lactis, Corynebacterium glutamicum, or yeast, with Escherichia coli BL21DE3 being preferred.

[0012] Preferably, the recombinant bacteria is recombinant Escherichia coli.

[0013] The present invention also provides a method for preparing the engineered bacteria that produce the amidase, characterized by comprising the following steps:

[0014] (1) The whole gene nucleotide sequence of the amidase gene apuh, as shown in SEQ ID NO.2, is synthesized; (2) The amidase gene apuh is ligated into the vector pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-apuh; (3) The recombinant plasmid obtained in step 2 is introduced into Escherichia coli BL21DE3 to obtain recombinant Escherichia coli BL21DE3 (pRSFDuet-apuh), and the expression of the apuh gene in the recombinant bacteria is regulated by the T7 promoter; (4) The recombinant bacteria BL21DE3 (pRSFDuet-apuh) is used to ferment and produce amidase with ethyl carbamate hydrolase activity.

[0015] Fermentation by recombinant bacteria can be achieved by culturing E. coli BL21DE3(pRSFDuet-apuh) genetically engineered bacteria to a certain cell concentration (e.g., OD). 600 =0.6-0.8), add an inducer (such as IPTG with a final concentration of 1mM), and culture for a certain period of time (such as 10-30h) to efficiently express amidase that can degrade ethyl carbamate.

[0016] The present invention also provides the application of the high-salt-resistant ammonase, the recombinant expression vector, and the recombinant Escherichia coli in the degradation of ethyl carbamate.

[0017] The application is the degradation of ethyl carbamate in fermented foods.

[0018] The fermented foods include soy sauce, vinegar, and pickled vegetables.

[0019] The application is the degradation of ethyl carbamate in alcoholic beverages.

[0020] The beneficial effects of this invention are as follows: The amidase obtained by this invention has the ability to degrade ethyl carbamate. It also exhibits good stability and catalytic activity under conditions containing high salt (NaCl, 10-20%, w / v) or high concentrations of ethanol (10-20%, v / v) and acidity, which facilitates the efficient application of this enzyme in the degradation of ethyl carbamate in high-salt fermented foods and alcoholic beverages. When expressed in *E. coli*, the recombinant bacteria, after fermentation and cell disruption, showed an enzyme activity of 30.1 U / mL with ethyl carbamate as a substrate. After purification by Ni-NTA affinity chromatography, the specific enzyme activity with ethyl carbamate as a substrate was 284.1 U / mg. This enzyme exhibits extremely high salt tolerance and good ethanol tolerance. It retains 100% enzyme activity in the presence of 10% NaCl and 65% activity in the presence of 15% NaCl. Furthermore, it retains 85%, 58%, 30%, and 20% activity in 10%, 20%, 30%, and 40% (v / v) ethanol, respectively. Even after exposure to less than 15% (v / v) ethanol and incubation at 37°C for 1 hour, it retains 78% or higher of its activity, demonstrating high stability in ethanol-containing environments. The recombinant enzyme prepared in this invention lays the foundation for the reduction of ethyl carbamate in high-salt traditional fermented foods and alcoholic beverages and for the industrial production of ethyl carbamate hydrolases, yielding significant economic and social benefits. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the construction of the expression plasmid pRSFDuet-apuh of this invention;

[0023] Figure 2This is an SDS-PAGE protein electrophoresis image of the expression product of recombinant E. coli BL21(DE3) / pRSFDuet-apuh in this invention; where 1 is the supernatant sample of cell lysate after induction of recombinant E. coli BL21(DE3) / pRSFDuet-apuh (1a, 1b are parallel samples), and 2 is the control (whole cell sample of recombinant bacteria without apuh gene).

[0024] Figure 3 This is a diagram of the composite structure of the amidase ApUH and EC after molecular docking; the APUH structure is obtained by homology modeling.

[0025] Figure 4 These are substrate-specific colorimetric images of the recombinant amidase of the present invention;

[0026] Figure 5 This is the salt tolerance curve of the recombinant amidase ApUH of the present invention;

[0027] Figure 6 Ethanol tolerance curve of recombinant amidase ApUH of the present invention; (A) relative enzyme activity of ApUH under different ethanol contents, (B) stability of ApUH at different ethanol concentrations;

[0028] Figure 7 The optimal pH and pH stability curves of the recombinant amidase ApUH of this invention are shown; (A) optimal pH, (B) pH stability.

[0029] Figure 8 The optimal temperature and temperature stability curves of the recombinant amidase ApUH of the present invention are shown; (A) optimal temperature, (B) temperature stability. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0031] It should be noted that, unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0032] In this invention, the LB medium used contains: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride (1.5% agar powder is added to the fixation medium).

[0033] The following commercially available plasmids and E. coli are used for gene cloning and expression:

[0034] pRSFDuet-1 (Novagen, USA)

[0035] E. coli BL21(DE3) (Novagen, USA)

[0036] Method for determining the activity of ethyl carbamate hydrolase: Take two 10 mL colorimetric tubes and add 1 mL of enzyme solution and 1 mL of inactivated enzyme solution to each tube. Then add 1 mL of 3% EC substrate solution (prepared with 20 mM pH 7.0 phosphate buffer) to each tube. After reacting in a 37℃ water bath for 30 min, add 1 mL of stop agent (10% trichloroacetic acid) to each tube, mix well, and then add 1 mL of colorimetric reagent I (15 g phenol and 0.625 g sodium nitrosoferricyanide diluted to 250 mL with ultrapure water) and 1 mL 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℃ water bath for 20 min. Remove from the bath, dilute with ultrapure water to 10 mL, and measure the OD value at 625 nm. Calculate the enzyme activity (using ammonium chloride to plot a standard curve).

[0037] The enzyme activity unit of ethyl carbamate hydrolase is defined as the amount of ethyl carbamate produced per minute by degradation of 1 μmol NH4 under normal pressure, 20 mM pH 7.0 phosphate buffer, and 37°C. + The required amount of enzyme is one unit of enzyme activity.

[0038] Example 1

[0039] Construction of recombinant expression strains of amidase

[0040] The process of constructing recombinant expression vectors is as follows: Figure 1 As 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 *Alicyclobacillus pomorum* (GeneBank Accession number: WP_026964413.1) in NCBI, the codons of the amidase encoding gene (named apuh in this invention) were optimized using the *E. coli* expression system. The optimized gene sequence is shown in SEQ ID NO. 2. The amidase APUH in this invention has a maximum amino acid sequence similarity of no more than 52% with other currently reported enzymes with EC degradation activity, thus possessing novelty.

[0041] The synthesized gene was digested with Nco I and BamHI, and after gel extraction and recovery, ligated to the pRSFDuet-1 plasmid digested with Nco I and BamHI. 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 (SEQ ID NO. 3) and reverse primer R (SEQ ID NO. 4). (Amplification system: Prepare a 20 μl reaction system according to the 2×tag kit instructions. Amplification conditions: 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, 72°C for 10 min per cycle.) Positive clones that were 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. The plasmid, verified to be correct by double digestion with Nco I and BamHI, was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. This yielded the exogenous expression system BL21(DE3) / pRSFDuet-apuh for recombinant ethyl carbamate hydrolase.

[0042] Forward primer F: 5'-CCATGGCAAAAAGCCCGACCAAAGC-3'

[0043] Reverse primer R: 5'-CAGAACCGTCATCACCACCACCACCACTAAGGATCC-3'

[0044] Recombinant expression and purification of amidases

[0045] Single colonies of the genetically engineered bacterium BL21(DE3) / pRSFDuet-apuh were picked and inoculated into 25 mL of LB broth containing 50 μg / mL kanamycin, and cultured overnight at 37°C with shaking. The next day, a 1% inoculum was transferred to TB broth containing 50 μg / mL kanamycin and cultured until the bacterial concentration reached OD500. 600 When the concentration of α=0.6, IPTG was added to a final concentration of 1 mmol / L for induction, and the cells were cultured at 30℃ for 10 h. The cells were collected by centrifugation, resuspended in 20 mM pH 7.4 phosphate buffer, and the cell walls were disrupted by sonication. The supernatant was collected by centrifugation, and the protein content and EC hydrolase activity in the supernatant were measured. Protein expression was detected by SDS-PAGE. Figure 2 The results showed that the enzyme activity of the crude enzyme solution after the recombinant cells were broken was 30.1 U / mL.

[0046] 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. The specific enzyme activity of the purified enzyme reached 284.1 U / mg.

[0047] Homology modeling and molecular docking of the amidase ApUH with EC molecules were performed using the online websites I-TASSER (https: / / zhanggroup.org / I-TASSER / ) and BSP-SLIM (https: / / zhanggroup.org / BSP-SLIM / ), respectively, resulting in the formation of the APUH-EC complex. Figure 3 This indicates that EC 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.

[0048] substrate specificity of recombinant amidases

[0049] The purified ApUH enzyme solution obtained above was mixed with EC and urea respectively, and the corresponding enzyme activities were determined according to the enzyme activity determination method described in the Materials and Methods section. The enzyme activity against EC was taken as 100%, and the relative enzyme activities of APUH against other substrates were calculated. The colorimetric images for enzyme activity detection are shown below. Figure 4 As shown, this amidase has no hydrolytic activity against urea, thus effectively avoiding substrate competition between urea and EC during enzyme application.

[0050] Salt tolerance of recombinant amidases

[0051] The purified enzyme ApUH obtained above was added to phosphate buffer (20 mM, pH 7.0) containing 3% EC and different mass concentrations (0-20%, 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%. The enzyme activity detection results are as follows: Figure 5 As shown. Figure 5It was found that at salt concentrations (0-5%), NaCl actually promoted the activity of this enzyme; under conditions containing 5% NaCl, the relative activity of the enzyme was 130%. This enzyme exhibits extremely high salt tolerance, maintaining 100% activity under conditions containing 10% NaCl and still retaining 65% activity under conditions containing 15% NaCl. These properties are far superior to those of currently reported EC hydrolases. In conclusion, this enzyme possesses extremely high salt tolerance and has the potential for application in the degradation of EC in traditional fermented foods (such as soy sauce).

[0052] Ethanol tolerance of recombinant amidases

[0053] The enzyme activity of ApUH 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 6 As shown in A, the enzyme still retains 85%, 58%, 30%, and 20% enzyme activity in ethanol containing 10%, 20%, 30%, and 40% (v / v), respectively.

[0054] ApUH was added to phosphate buffer (20 mM, pH 7.4) 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 6 As shown in B, the enzyme can still retain 78% or more of its activity after being placed in less than 15% (v / v) ethanol at 37°C for 1 hour, and it has high stability in an environment containing ethanol.

[0055] The ethanol tolerance assay results of ApUH indicate that the enzyme has high ethanol tolerance and has the potential to be applied to the degradation of EC in alcoholic beverages.

[0056] Optimal pH and pH stability of recombinant amidase

[0057] The enzyme activity of ApUH was measured under the following conditions: pH 3.0–8.0 (20 mM citrate-disodium hydrogen phosphate), pH 8.0–9.0 (20 mM Tris-HCl), and pH 9.0–10.0 (20 mM glycine-sodium hydroxide). The highest enzyme activity obtained was defined 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 7 As shown in A, the optimal pH for this enzyme is 7.0.

[0058] To determine pH stability, the purified amidase was added to buffer solutions of different pH values ​​(pH range 3.0-10.0) 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 7 As shown in B, the enzyme is relatively stable between pH 6.0 and 8.0, and can still maintain more than 80% of its enzyme activity after treatment; at the same time, after being placed at pH 4.0-5.0 for 6 hours, the enzyme can maintain about 20%-58% of its enzyme activity, and has a certain degree of acid tolerance.

[0059] The results of the optimal pH and pH stability determination of APUH indicate that this amidase is suitable for the degradation of EC in traditional fermented foods in weakly acidic, neutral and weakly alkaline conditions.

[0060] Optimal temperature and temperature stability of recombinant amidase

[0061] The activity of ApUH was measured at 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 °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 60℃.

[0062] APUH enzyme solutions were incubated at 20, 30, 40, 50, 60, 70, and 80°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 100% of its activity after incubation at 50°C or lower for 30 minutes. However, its thermal stability decreases sharply at temperatures above 50°C.

[0063] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, 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 variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. The use of a high-salt-tolerant amidase, a recombinant expression vector containing a gene encoding the high-salt-tolerant amidase, and a recombinant bacterium containing the recombinant expression vector in the degradation of ethyl carbamate, wherein the amino acid sequence of the amidase is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The recombinant bacteria are any one of recombinant Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, Lactococcus lactis, Corynebacterium glutamicum, or yeast.

3. The application according to claim 2, characterized in that, The Escherichia coli is Escherichia coli BL21(DE3).

4. The application according to claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

5. The application according to claim 1, characterized in that, The application is the degradation of ethyl carbamate in fermented foods.

6. The application according to claim 5, characterized in that, The fermented foods include soy sauce, vinegar, and pickled vegetables.

7. The application according to claim 1, characterized in that, The application is the degradation of ethyl carbamate in alcoholic beverages.

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