Acid-resistant and heat-resistant nitrilase mutant and application thereof in fermentation of rapeseed meal

Abstract

The invention belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to an acid-resistant and heat-resistant nitrilase mutant and application thereof in fermentation of rapeseed meal. Aiming at the conditions that the existing nitrilase is insufficient in acid resistance and heat resistance and relatively low in activity, the nitrilase RsNit anchored on the surface capsid protein cotG of bacillus subtilis DB403 is used as a starting target for modification; according to the nitrilase mutant disclosed by the invention, a catalytic hot spot existing in an active center is found by carrying out semi-rational design on amino acid at the active center, and combined mutation is carried out on the catalytic hot spot, so that it is found for the first time that RsNit has a catalytic hot spot on 4-pentenenitrile at the active center, and a nitrilase mutant of which the activity, the thermal stability and the acid tolerance are all remarkably improved is successfully screened out; the method is applied to fermentation of the rapeseed meal, so that full utilization of the rapeseed meal in livestock and poultry feed is promoted, and the method has a wide market application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to an acid-tolerant and heat-tolerant nitrilase mutant and application thereof in fermented rapeseed meal. BACKGROUND

[0002] In recent years, the huge demand for rapeseed oil has led to a corresponding high yield of rapeseed oil by-product, meal, making rapeseed meal the second largest traded protein ingredient after soybean meal (USDA 2017) and an important protein source. However, glucosinolates (GLS) remaining in rapeseed meal are major anti-nutritional factors, which can produce toxic compounds such as isothiocyanates, thiocyanates, nitriles, thiocyanates, oxazolidine sulfones, etc. through endogenous or exogenous conversion, thereby causing liver, kidney and thyroid damage. It is worth mentioning that the products of glucosinolate degradation also have preferences under different pH conditions, among which, under low pH (pH 2-5) conditions, glucosinolates are mainly converted into nitriles, which have greater carcinogenicity and mutagenicity than other secondary metabolites. Currently, the main rapeseed comes from Brassica juncea, Brassica napus, Brassica campestris, Brassica carinata, among which 3-butylthioglucoside, 4-pentenyl glucosinolate, 2-hydroxy-3-butylthioglucoside, and 2-propenyl glucosinolate are the most common types of glucosinolate in these rapeseed. They can be degraded into 4-pentenyl nitrile, 5-hexenyl nitrile, 3-hydroxy-pent-4-enyl nitrile, and 3-butyl nitrile, respectively, under acidic conditions such as fermentation process or animal gastrointestinal tract. Therefore, in order to improve the utilization rate and nutritional value of rapeseed meal, it is necessary to screen nitrilases that can efficiently degrade the main nitrile substances in rapeseed meal.

[0003] Nitrile hydrolases can hydrolyze nitrile into the corresponding carboxylic acids and ammonia, thus detoxifying rapeseed meal and greatly promoting the conversion and degradation of nitrile substances in it. However, natural nitrile hydrolases are difficult to meet the needs of the feed industry. For example, the feed pelleting process requires high temperatures of 60-90℃, which necessitates that the feed enzymes have a certain degree of heat resistance. Furthermore, lactic acid bacteria and enzyme fermentation of feed is currently a popular field. Adding lactic acid bacteria and yeast during the fermentation of rapeseed meal can reduce the content of anti-nutritional factors in the meal, requiring the added feed enzymes to have strong acid resistance. Since the animal stomach is an acidic environment, this means that if the feed enzymes have acid resistance, they can continue to function in the animal's stomach. Current research on nitrile hydrolases mainly focuses on exploring the factors affecting substrate specificity, such as the study by Shubhangi Kaushik et al. on the effect of site-directed mutagenesis on substrate specificity. Other research focuses on analyzing the reasons for the increased enzyme activity in mutants, such as the molecular simulation study by Luo Zhijian of Jilin University on the increased activity of nitrile hydrolases from Synechocystis sp. PCC6803. Some researchers have also explored the mechanism of nitrile hydrolase thermal stability. For example, Tang Xiaomang of East China University of Science and Technology used computational methods to explore the contribution of electrostatic distribution on the protein surface to protein thermal stability. He proposed the hypothesis that optimizing the electrostatic interaction on the surface will benefit the thermal stability of the protein, and proved it by designing mutations.

[0004] However, nitrile hydrolases exhibit stronger activity under neutral to alkaline conditions and lower activity under acidic conditions. Research on acid-resistance modification is limited, and the mechanisms underlying their acid resistance remain unclear. Currently, commercially available nitrile hydrolases are only suitable for neutral or alkaline conditions. Therefore, modifying nitrile hydrolases to improve their acid resistance or heat resistance, and promoting their application in the feed and aquaculture industries, is of significant research value. Summary of the Invention

[0005] This invention addresses the shortcomings of existing nitrile hydrolases, such as insufficient acid and heat resistance and low activity. It modifies a nitrile hydrolase, RsNit, anchored to the capsid protein cotG on the surface of Bacillus subtilis DB403. Through semi-rational design of the amino acids at the active site, this invention identifies catalytic hotspots and performs combined mutations on these hotspots. This successfully screened a nitrile hydrolase mutant with significantly improved activity, thermal stability, and acid tolerance. Its application in rapeseed meal fermentation has yielded unexpected technical results, promoting the full utilization of rapeseed meal in livestock and poultry feed.

[0006] To achieve the above objectives, the present invention provides the following technical solutions;

[0007] (a) An acid- and heat-resistant nitrile hydrolase mutant;

[0008] The nitrile hydrolase mutant T1 is obtained by mutating valine (Val) at position 190 to leucine (Leu) and phenylalanine (Phe) at position 194 to histidine (His) in the nitrile hydrolase RsNit, whose amino acid sequence is shown in SEQ ID NO.2; wherein, the nucleotide sequence of the nitrile hydrolase RsNit is shown in ID NO.1; and the amino acid sequence of the acid-resistant and heat-resistant nitrile hydrolase mutant is shown in SEQ ID NO.6.

[0009] The method for obtaining the nitrile hydrolase mutant is as follows:

[0010] (1) The nitrile hydrolase gene sequence (GenBank: JN635494) from Rhodobacter sphaeroides LHS-305 was optimized according to the codon preference in Bacillus subtilis. The nucleotide sequence of the codon-optimized RsNit gene is shown in SEQ ID NO.1.

[0011] Using the optimized RsNit gene as a template, PCR amplification was performed to obtain the amplified RsNit gene fragment. The shuttle expression vector pHS-cotG and the RsNit gene fragment were digested with restriction endonucleases Spe I and HindIII. Then, the digested linearized plasmid vector pHS-cotG and the RsNit gene fragment were ligated together using T4 DNALigase and transformed into E. coli DH5α competent cells to obtain the recombinant shuttle plasmid DH5α-pHS-cotG-RsNit.

[0012] (2) Using the recombinant plasmid DH5α-pHS-cotG-RsNit described in step (1) as a template, amplification was performed using the mutant primers V190L-F and V190L-R to obtain a full plasmid PCR product containing the V190L mutation; then, the full plasmid PCR product containing the V190L mutation was treated with the restriction endonuclease Dpn I, and the original template was selectively digested. The full plasmid PCR product containing the V190L mutation treated with DpnI was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pHS-cotG-RsNit-V190L;

[0013] Preferably, the primers for site-directed mutagenesis of the whole plasmid PCR in step (2) are the two primers of V190L, with the following sequences:

[0014] V190L-F: CTGCTGGGTCCGATTTTTGGTGAACAGATT,

[0015] V190L-R:ACCCAGCAGTGAACCCGGAAAATGGCTC;

[0016] The reaction system for site-directed mutagenesis of whole plasmid PCR is as follows: 1 μL each of 10 μM V190L-F and V190L-R primers, 10 μL of 2×PrimeSTARMax Premix, 1 μL of DH5α-pHS-cotG-RsNit-V190L plasmid template (concentration 0.1-1 ng / μL), and ddH2O to make up to 20 μL.

[0017] The amplification program for whole plasmid PCR was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min 10 s (30 cycles in total); 72℃ final extension for 10 min.

[0018] (3) Using the mutant plasmid DH5α-pHS-cotG-V190L obtained in step (2) as a template, full plasmid PCR was performed using F194H mutant primers to amplify the PCR product containing the F194H mutation; the PCR product containing the F194H mutation was treated with restriction endonuclease DpnI to selectively digest the original template plasmid; the PCR product containing the F194H mutation treated with DpnI was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pHS-cotG-RsNit-V190L-F194H, denoted as DH5α-pHS-cotG-RsNit-T1; the nitrile hydrolase mutant gene fragment carried by the mutant plasmid was denoted as RsNit-T1.

[0019] Preferably, the sequences of the F194H mutation primers (F194H-F and F194H-R) in step (3) are as follows:

[0020] F194H-F;GGTCCGATTCATGGTGAACAGATTGAAGTT

[0021] F194H-R: CTGTTCACCATGAATCGGACCAACCAGTGA

[0022] The whole plasmid PCR amplification system is as follows: 1 μL of 10 μM F194H-F primer, 1 μL of 10 μM F194H-R primer, 10 μL of 2×PrimeSTARMax Premix, 1 μL of template plasmid DH5α-pHS-cotG-RsNit-V190L (concentration of 0.1-1 ng / μL), and ddH2O to make up to 20 μL.

[0023] Whole plasmid PCR amplification program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min 10 s (30 cycles in total); 72℃ final extension for 10 min.

[0024] (ii) A gene encoding the acid- and heat-resistant nitrile hydrolase mutant: the nucleotide sequence of the gene is shown in SEQ ID NO.5.

[0025] (iii) An expression vector: the expression vector (pHS-cotG-RsNit-T1) carries a gene encoding a nitrile hydrolase mutant.

[0026] (iv) Construction of engineered bacteria expressing nitrile hydrolase mutants: The engineered bacteria expressing nitrile hydrolase mutants are obtained by transforming the expression vector into host cells;

[0027] The specific steps are as follows: using Bacillus subtilis DB403 as the host bacterium, a mutant plasmid (DH5α-pHS-cotG-RsNit-T1) is introduced through chemical transformation to obtain the engineered bacterium B. subtilis DB403-pHS-cotG-RsNit-T1.

[0028] (V) Preparation of the mutant enzyme preparation cotG-RsNit-T1:

[0029] The engineered bacterium B. subtilis DB403-pHS-cotG-RsNit-T1 was inoculated into DSM medium and cultured. After culture, it was centrifuged to obtain a recombinant spore suspension displaying nitrile hydrolase on the surface; or the spore suspension was freeze-dried to prepare a solid powder. The recombinant spore suspension or solid powder is a mutant enzyme preparation, denoted as cotG-RsNit-T1.

[0030] The specific steps are as follows: The engineered bacterium *B. subtilis* DB403-pHS-cotG-RsNit-T1 is inoculated into LB medium and cultured for the first time. The culture is then inoculated into Difco spore-forming medium (DSM medium), and after a second culture, the bacterial precipitate is collected by centrifugation. The precipitate is resuspended in GTE buffer containing lysozyme. The resuspended precipitate is incubated, centrifuged again, and washed with PBS buffer. The precipitate is then resuspended in PBS buffer to obtain a recombinant spore suspension exhibiting nitrile hydrolase on its surface. Alternatively, the recombinant spore suspension can be freeze-dried to obtain a solid powder. Both the recombinant spore suspension and the solid powder are mutant enzyme preparations, denoted as cotG-RsNit-T1.

[0031] Preferably, the concentration of lysozyme in the GTE Buffer is 2 mg / mL, wherein the components of the GTE Buffer are: 10 mM EDTA, 20 mM Tris-HCl (pH = 7.5), and 50 mM Glucose.

[0032] The first culture conditions were 37℃, 180 rpm for 12 h; the second culture conditions were 37℃, 180 rpm for 36 h; the GTE buffer composition was: 50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL, pH 7.5; the incubation conditions were: 37℃ for 45 min; the OD of the recombinant spore protein suspension was... 600 The value is 1.

[0033] (vi) Application of nitrile hydrolase preparations in fermented rapeseed meal:

[0034] The feed ingredients consist only of rapeseed meal; lactic acid bacteria are first inoculated into the rapeseed meal, and then calcium is added. 2+ A fermentation system was obtained by combining the mutant enzyme preparation (cotG-RsNit-T1), and the water content was controlled during fermentation to achieve the application of degrading glucosinolates in fermented rapeseed meal.

[0035] Based on the percentage of feed ingredients by weight, the lactic acid bacteria is *Lactobacillus plantarum*, added at 10%; the mutant enzyme preparation added at 10%; and Ca... 2+ The addition amount is 2%, the total moisture content of the feed is 40% to 45%; the fermentation temperature is 25-40℃, and the fermentation time is 3-7 days.

[0036] The advantages and technical effects of this invention are:

[0037] 1. High-efficiency production of immobilized nitrile hydrolase preparations: This invention successfully achieves a simple and rapid production of immobilized nitrile hydrolase preparations using a Bacillus subtilis spore surface display system. This method not only increases the yield of enzyme preparations but also yields enzyme products with excellent stability and recyclability, providing strong technical support for industrial production.

[0038] 2. Semi-rational design of catalytic hotspots for the active site of nitrile hydrolase based on spore display high-throughput screening platform: The present invention first uses an alanine scanning strategy to perform the first round of mutations on the active site of nitrile hydrolase, then selects key sites and performs the first round of saturation mutations in combination with high-throughput screening, and then determines the catalytic hotspot amino acids of the active site through saturation mutations to perform the second round of combined saturation mutations, thereby obtaining a nitrile hydrolase mutant with significantly improved activity.

[0039] 3. Application in fermented rapeseed meal: The nitrile hydrolase immobilized enzyme preparation prepared in this invention has shown excellent performance in the fermentation of rapeseed meal. Experiments have proven that this enzyme preparation can effectively improve the degradation rate of glucosinolates in rapeseed meal. Attached Figure Description

[0040] Figure 1 Agarose gel electrophoresis image of the PCR amplification fragment of the nitrile hydrolase gene.

[0041] Figure 2 : Double digestion of nitrile hydrolase gene (left image) and plasmid pHS-cotG (right image).

[0042] Figure 3 Western blot verification of recombinant nitrile hydrolase after display on the surface of recombinant engineered B. subtilis DB403 pHS-cotG-RsNit spores.

[0043] Figure 4 Immunofluorescence verification images of recombinant nitrile hydrolase after display on the surface of recombinant engineered strain B. subtilis DB403 pHS-cotG-RsNit spores; where No. 1 is the immunofluorescence white light field of B. subtilis DB403 pHS-cotG spores, No. 2 is the immunofluorescence fluorescence field of B. subtilis DB403 pHS-cotG spores, No. 3 is the immunofluorescence white light field of B. subtilis DB403 pHS-cotG-RsNit spores, and No. 4 is the immunofluorescence fluorescence field of B. subtilis DB403 pHS-cotG-RsNit spores.

[0044] Figure 5 The effects of different metal ion concentrations and types on immobilized recombinant spore nitrile hydrolase.

[0045] Figure 6 Active center substrate Amino acids within the specified range.

[0046] Figure 7 Substrate The relative enzyme activity of alanine within the range of amino acids.

[0047] Figure 8 : The interaction force between the substrate and the amino acid at the active site.

[0048] Figure 9 : Relative enzyme activity due to saturation mutation of key amino acids in the active site.

[0049] Figure 10 : Relative enzyme activity of catalytic hotspot combination saturation mutation.

[0050] Figure 11 Molecular docking analysis of interactions between wild-type RsNit and its mutants and substrates; Figure A shows the interaction force analysis of wild-type RsNit, Figure B shows the interaction force analysis of mutant T1, Figure C shows the interaction force analysis of mutant T2, Figure D shows the interaction force analysis of mutant T3, and Figure E shows the interaction force analysis of mutant T4.

[0051] Figure 12 Molecular dynamics simulations of wild-type RsNit and mutants T1 and T4: RMSD comparison, RMSF comparison, and SASA comparison after MD simulation (a), (b), and (c), respectively.

[0052] Figure 13 Enzymatic properties analysis of mutant T1: optimal pH (a), optimal temperature (b), pH tolerance (c), and temperature tolerance (d).

[0053] Figure 14 : Standard curve of glucosinolates.

[0054] Figure 15 The effect of adding nitrile hydrolase preparation on glucosinolate content in rapeseed meal. Detailed Implementation

[0055] The implementation process of this invention is not limited to what is described below. While maintaining its basic principles and effects, corresponding adjustments and improvements can be made as needed. Therefore, the scope of protection of this invention should not be limited to the specific descriptions of the following embodiments. All technical solutions and improvements that do not depart from the spirit and scope of this invention should be covered within the scope of the claims of this invention. Unless otherwise specified, the experimental materials mentioned in the embodiments can be purchased through market channels. Regarding experimental methods and operating procedures, unless otherwise specified, they follow existing mature technologies and general specifications in the field.

[0056] Reagent and material specifications:

[0057] The nitrile hydrolase gene RsNit (GenBank: JN635494) from the genus *Rhodobacter phaeroides* LHS-305 was synthesized with codon optimization by General Biotech (Chuzhou) Co., Ltd., and inserted into the pET-28a plasmid. 3-Butenonitrile, 4-pentenonitrile, 5-hexenonitrile, benzonitrile, and 3-hydroxy-4-pentenonitrile were purchased from Aladdin Industries, Inc. (Shanghai, China). MgCl2, KCl, BaCl2, CuSO4, CaCl2, FeCl3, NiCl, FeCl2, MnCl2, and ZnCl2 were purchased from Sinopharm Group. Restriction endonucleases Spe I and HindIII, and T4 DNA Ligase were purchased from Takara Biotech (Beijing) Co., Ltd. The recipient bacterium *Escherichia coli* DH5α competent cells were purchased from Sangon Biotech Co., Ltd.

[0058] Obtaining the fermentation seed culture: Lactic acid bacteria were inoculated into MRS medium at a 1% inoculum and cultured anaerobically at 37°C until OD500. 600nm =0.8-1.0, which yields the fermented seed liquid.

[0059] The following embodiments relate to a method for determining the activity of nitrile hydrolase using 4-pentenonitrile as a substrate, and the specific operation is as follows:

[0060] Add 10 mM nitrile substrate to a 1 mL reaction system. Use PBS (pH 7) as the reaction buffer. After adding the enzyme solution, incubate the reaction at 37°C for 1 h. Then, terminate the reaction with 100 μL of 2.0 M hydrochloric acid. Transfer 100 μL of the product from the reaction system to a 10 mL test tube. Add 1 mL of sodium phenolate, 1.5 mL of sodium nitroferricyanide, and 1.5 mL of sodium hypochlorite sequentially. Make up to 5 mL with deammonium chloride solution, invert and mix thoroughly. Incubate at 37°C in a metal water bath for 15 min. Transfer 200 μL to a 96-well microplate and read the OD value using a microplate reader. 630 The absorbance was measured at the point.

[0061] Example 1: Immobilization preparation method of recombinant nitrile hydrolase:

[0062] Construction of recombinant nitrile hydrolase engineered bacteria:

[0063] First, the nitrile hydrolase gene sequence from Rhodobactersphaeroides LHS-305 (GenBank: JN635494) was retrieved from the NCBI database, and its encoded amino acid sequence is shown in SEQ ID NO.2. Then, the gene sequence was optimized according to the codon preference in Bacillus subtilis, and the nucleotide sequence of the codon-optimized RsNit gene is shown in SEQ ID NO.1.

[0064] To amplify the target gene sequence, forward primers (SEQ ID NO.3) and reverse primers (SEQ ID NO.4) with restriction enzyme sites were designed. The RsNit gene was obtained after PCR amplification. The linearized fragment of the target gene is shown below. Figure 1 The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 3 min; 30 cycles of 94℃ for 15 s, 55℃ for 15 s, and 72℃ for 1 min; and extension at 72℃ for 10 min.

[0065] The shuttle expression vector pHS-cotG and the RsNit gene were digested with restriction endonucleases Spe I and HindIII, and the linearized target fragment was recovered and the product concentration was determined. The linearized plasmid vector pHS-cotG and the RsNit gene fragment were ligated together using T4 DNA Ligase, and then transformed into E. coli DH5α competent cells. Positive transformants were selected, and the recombinant plasmid DH5α-pHS-cotG-RsNit was successfully obtained. The construction process is described in [link to documentation]. Figure 2 .

[0066] The recombinant shuttle plasmid DH5α-pHS-cotG-RsNit was transformed into B. subtilis DB403 competent cells. Positive transformants were detected by colony PCR, and the recombinant nitrile hydrolase engineered strain B. subtilis DB403-pHS-cotG-RsNit was obtained.

[0067] Meanwhile, the recombinant shuttle expression vector pHS-cotG was treated in the same way, that is, the recombinant shuttle plasmid pHS-cotG was directly transformed into B. subtilis DB403 competent cells, and the positive transformants were detected by colony PCR to obtain the recombinant engineered strain B. subtilis DB403-pHS-cotG, which served as a blank control.

[0068] Identification of expression in recombinant engineered strain B. subtilis DB403-pHS-cotG-RsNit:

[0069] S1. Transformants were picked from plates and verified using P1-F and PR primers to detect single colonies of *B. subtilis* DB403-pHS-cotG-RsNit. PCR reaction conditions were: pre-denaturation at 94℃ for 3 min; intra-cycle PCR: 94℃ for 15 s, 55℃ for 15 s, 72℃ for 1 min, for a total of 30 cycles; followed by extension at 72℃ for 10 min. P1-F: TGGCCATGGCACCGAATCTGGAT, P1-R: GTGCTCGAGATTCGGTGACGGC.

[0070] The verified Bacillus subtilis DB403 transformants were inoculated at a rate of 1% in 5 mL LB medium containing 7.5 μg / mL chloramphenicol and cultured overnight at 37°C and 170 rpm. Then, 1% of the bacterial suspension was inoculated into 100 mL of Difco Sporulation Medium (DSM) and cultured at 37°C and 170 rpm for 40 h. After 40 h of culture, the supernatant was removed by centrifugation at 8000 rpm for 10 min, and the bacterial pellet was collected. GTE buffer was added, and the pellet was incubated at 37°C for 30 min to lyse the vegetative cells. The spores were then recovered by centrifugation and washed twice with PBS (4000 rpm, 10 min). Finally, the spores were resuspended in PBS buffer, and the OD of the spore suspension was adjusted. 600 The concentration is 1.0, denoted as cotG-RsNit spore solution, for later use.

[0071] S2. Treat the control engineered bacterium B. subtilis DB403-pHS-cotG according to the method in S1, except that B. subtilis DB403-pHS-cotG-RsNit is replaced with B. subtilis DB403-pHS-cotG. The final spore suspension is denoted as cotG empty spore solution.

[0072] To verify the display of nitrile hydrolase RsNit on the spore surface, the obtained spore suspension was centrifuged and resuspended in an equal volume of decoating extraction buffer (1% SDS, 50 mM dithiothreitol), and treated at 70°C for 1 h to obtain the cotG-RsNit fusion protein. The extracted protein was verified by Western blotting. Since the target gene carries a 6×His tag, the primary antibody was Anti-6×His Tag mouse monoclonal antibody, and the secondary antibody was HRP-conjugated-affinipure Goat Anti-Mouse-IgG. Recombinant spores containing empty plasmids were simultaneously treated as a control, and the results were observed using an Omni-ECL™ alkaline chemiluminescence kit (Epizyme Biotech). The results are as follows: Figure 3 As shown, this indicates that the recombinant nitrile hydrolase immobilized enzyme preparation cotG-RsNit has been successfully displayed on the surface of Bacillus subtilis spores.

[0073] Glass slides were soaked in 0.1% (w / v) gelatin overnight. Then, 10 μL of cotG-RsNit spore solution was spread evenly on the slides and air-dried at room temperature. The slides were then rinsed three times with PBS, 5 min each time. The slides were then placed in PBS containing 5% (w / v) skim milk powder and blocked at room temperature for 30 min, followed by rinsing three times with PBS, 5 min each time. The slides were incubated with the primary antibody Anti-6×His Tagmouse monoclonal-antibody overnight at 4°C, then washed three times with PBS, 5 min each time. Finally, the slides were incubated with a 100-fold diluted fluorescent secondary antibody GoatAnti-Mouse IgG / FITC in the dark for 2 h, followed by washing again, 5 min each time. The empty plasmid recombinant spore solution (cotG) was simultaneously treated as a control. After drying, the differences between the experimental and control groups were observed using a fluorescence microscope. The results are as follows: Figure 4 As shown.

[0074] Effects of metal ions on recombinant engineered bacteria B. subtilis DB403-pHS-cotG-RsNit

[0075] Following the enzyme activity assay system, the reaction was carried out at 37℃ and pH=7 to investigate the effects of different ions on the immobilized enzyme cotG-RsNit. These mainly included Mg... 2+ K + Ba 2+ Cu 2+ Ca 2+ Fe 3+ Ni + Fe 2+ Mn 2+ Zn 2+ The effect of ion concentration on the immobilized enzyme cotG-RsNit was investigated using three concentrations: 1 mM, 3 mM, and 5 mM. Each ion assay was performed in triplicate, with at least three replicates. Results are as follows: Figure 5 As shown, 5mM Mg 2+ 1mM and 5mM Ca 2+ All of these can improve the activity of the immobilized enzyme cotG-RsNit to some extent. Especially when 5 mM Ca is added... 2+ At that time, the enzyme activity increased to 1.4 times the original level.

[0076] Example 2: High-throughput screening and semi-rational design of recombinant nitrile hydrolases

[0077] (1) Homology modeling of RsNit was performed using SWISS-MODEL. The model was then docked with the ligand 4-pentenonitrile in AutoDockVina software. The specific steps were: first, water molecules were removed from the protein and hydrogen was added to select it as the acceptor; then, the ligand was hydrogenated; the ligand and acceptor were docked multiple times, and the most reasonable docking conformation was selected as the result. Based on the position of the catalytic triplet and the results of molecular docking, a three-dimensional structure diagram of the active site was drawn using PyMOL software, and the ligand 4-pentenonitrile was selected as the center. Amino acids within the range serve as substrate binding pockets. amino acids within the range such as Figure 6 As shown.

[0078] (2) Based on the molecular docking results, a comparison was made with the active centers of Nit6803 with known crystal structures, expanding the distance to... To more comprehensively explore the interaction between the substrate and the amino acids in the binding pocket. The substrate... Fifteen amino acids within the range—Y51, F56, N110, T131, Y132, H133, E134, W138, W162, P186, L189, V190, I193, F194, and Q197—were mutated to alanine in order to study the mutation status at each point in isolation.

[0079] The primer sequences are as follows:

[0080] Y51A-F GCCGTGGGCGCCGTATTTTAGCTTTGTTC

[0081] Y51A-R AAATACGGCGCCCACGGCACAAAAGGTTTC

[0082] F56A-F AGCGCGGTTCTGCCGCCGGTTCAGCAG

[0083] F56A-R AACCGCGCTAAAATACGGATACCACGGCAC

[0084] N110A-F ACTGTATGCGACACAGCTGATTTTTGATGC

[0085] N110A-R CAGCTGTGTCGCATACAGTGAGCCATGATC

[0086] T131A-F ATTACACCGGCATATCATGAACGTATGATT

[0087] T131A-R TTCATGATATGCCGGTGTAATTTTACGACG

[0088] Y132A-F ACACCGACAGCGCATGAACGTATGATTTGG

[0089] Y132A-R CGTTCATGCGCTGTCGGTGTAATTTTACGA

[0090] H133A-F CCGACATATGCGGAACGTATGATTTGGGGT

[0091] H133A-R TACGTTCCGCATATGTCGGTGTAATTTTAC

[0092] E134A-F CATATCATGCACGTATGATTTGGGGTCAGG

[0093] E134A-R CATACGTGCATGATATGTCGGTGTAATTTT

[0094] W138A-F CGTATGATTGCGGGTCAGGGCGATGGTGCG

[0095] W138A-R GCCCTGACCCGCAATCATACGTTCATGATA

[0096] W162A-F TGCGCGGAACATTATAATCCGCTGGCGCG

[0097] W162A-R TATAATGTTCCGCGCACGCCAGTGCACCC

[0098] P186A-F AGCCATTTTGCGGGTTCACTGGTTGGTCCG

[0099] P186A-R GTGAACCCGCAAAATGGCTCGCATGAATTT

[0100] L189A-F TCAGCGGTTGGTCCGATTTTTGGTGAACAG

[0101] L189A-R AACCGCTGAACCCGGAAAATGGCTCGCA

[0102] V190A-F CTGGCGGGTCCGATTTTTGGTGAACAGATT

[0103] V190A-R ACCCGCCAGTGAACCCGGAAAATGGCTC

[0104] I193A-F GTTGGTCCGGCGTTTGGTGAACAGATTGAA

[0105] I193A-R TTCACCAAACGCCGGACCAACCAGTGAACC

[0106] F194A-F CCGATTGCCGGTGAACAGATTGAAGTTACA

[0107] F194A-R TGTTCACCGGCAATCGGACCAACCAGTGAA

[0108] Q197A-F TTGGTGAAGCGATTGAAGTTACAATGCGTC

[0109] Q197A-R ACTTCAATCGCTTCACCAAAAATCGGACCA

[0110] Enzyme activity was detected using 4-pentenonitrile as a substrate in phosphate buffer at 37°C and pH 7.0. The results of the alanine scanning experiment are as follows: Figure 7 As shown. Using the wild-type nitrile hydrolase RsNit activity as a control, mutants N110A, H133A, W138A, and P186A almost lost their enzyme activity, mutants Y51A, N110A, Y132A, E134A, and W162A maintained less than 80% of their enzyme activity, and mutants F56A, T131A, L189A, V190A, I193A, F194A, and Q197A maintained more than 80% of their enzyme activity. Among them, the enzyme activity of mutant T131A was slightly higher than that of the wild type, but there was no significant difference compared with the wild type.

[0111] (3) The interaction forces between the active centers before and after mutation were analyzed using Discovery Studio 2019 software, such as... Figure 8As shown, sites P186, L189, W162, V190, and F194 exhibit non-hydrogen bond interactions with the substrate 4-pentenonitrile. Furthermore, the alanine scan results indicate that T131, N110, H133, W138, and P186 are key amino acids affecting enzyme activity. Since Y51, H133, and E134 form a hydrogen bond network, they are not the preferred mutation sites. Therefore, the first round of saturation mutagenesis selected eight sites: T131, N110, W162, V190, F194, P186, L189, and W138. A spore display high-throughput screening method was used to screen for mutants, with the enzyme activity of wild-type RsNit serving as a positive control. OD 630 Mutants with higher activity than wild-type mutants indicate increased enzyme activity. At least two 96-well plates were used for screening at each site to ensure comprehensive mutant screening. Results are as follows: Figure 9 As shown in the figure, all mutants at the N110 site showed a significant decrease in enzyme activity after retesting, and all mutants at the T131 site showed no increase in enzyme activity, with decreases ranging from 51% to 88.1% compared to the wild type. Some mutants at the V190 and F194 sites showed decreased enzyme activity after retesting, such as the V190S mutant (43% decrease), V190D mutant (55% decrease), V190N mutant (20% decrease), and F194R mutant (42.2% decrease). Retesting revealed that some mutants at the V190 and F194 sites still maintained higher enzyme activities than the wild type. V190L showed a significant increase of 63.7% compared to the wild type, V190I a significant increase of 39.7%, F194T a significant increase of 14.4%, and F194D a significant increase of 26.3%.

[0112] (4) To obtain mutants with further enhanced enzyme activity, a saturation mutation was performed using the combination of V190 and F194. The primer sequences for the saturation mutation are shown below. High-throughput screening was conducted using a spore display high-throughput screening protocol, with wild-type RsNit as a positive control. To ensure comprehensive screening, six 96-well plates were screened for the saturation mutation. The mutants with enhanced enzyme activity from the initial screening were amplified and retested to ensure consistency with the OD values ​​of the wild-type RsNit spore solution. The results are as follows: Figure 10As shown in the figure, mutant T1 (V190L / F194H) showed the highest enzyme activity increase, reaching 243% of the pre-mutation level; followed by mutant T2 (V190S / F194Y), with activity increasing to 206% of the pre-mutation level. Furthermore, mutants T3 (V190R / F194T), T4 (V190T / F194S), and T5 (V190K / F194S) showed increases of approximately 70%, 60%, and 44% compared to the wild type, respectively. Mutant T6 (V190Y / F194R) showed an increase of approximately 16% compared to the wild type. Some of the combined mutants also showed a significant decrease in enzyme activity after retesting, such as mutants V190Y / F194G and V190G / F194H, which decreased to 36% and 16% of the wild type, respectively.

[0113] The primer sequences for saturation mutation are as follows:

[0114] N110-F GGCTCACTGTATNNKACACAGCTGATTTTT

[0115] N110-R ATACAGTGAGCCATGATCGCGTTCATTCAC

[0116] W138-F CGTATGATTNNKGGTCAGGGCGATGGTGCG

[0117] W138-R AATCATACGTTCATGATATGTCGGTGTAAT

[0118] W162-F GGTGCACTGGCGTGCNNKGAACATTATAAT

[0119] W162-RGCACGCCAGTGCACCCATGCGACCAACTTT

[0120] P186-F GCGAGCCATTTTNNKGGTTCACTGGTTGGT

[0121] P186-RAAAATGGCTCGCATGAATTTCTTCATGCTG

[0122] L189-F CCGGGTTCANNKGTTGGTCCGATTTTTGGT

[0123] L189-R TGAACCCGGAAAATGGCTCGCATGAATTTC

[0124] V190-F GGTTCACTGNNKGGTCCGATTTTTGGTGAA

[0125] V190-R CAGTGAACCCGGAAAATGGCTCGCATGAAT

[0126] I193-F GTTGGTCCGNNKTTTGGTGAACAGATTGAA

[0127] I193-R CGGACCAACCAGTGAACCCGGAAAATGGCT

[0128] F194-F GGTCCGATTNNKGGTGAACAGATTGAAGTT

[0129] F194-R AATCGGACCAACCAGTGAACCCGGAAAATG

[0130] T131-F ATTACACCGNNKTATCATGAACGTATGATT

[0131] T131-R CGGTGTAATTTTACGACGTTTCAGTTTCAG

[0132] V190-F194-F CGGGTTCACTGNNKGGTCCGATTNNKGGTGAACA

[0133] V190-F194-R CAGTGAACCCGGAAAATGGCTCGCATGAATTTC

[0134] The specific process for obtaining mutant T1 (V190L / F194H) is described below:

[0135] S1. Using the recombinant plasmid DH5α-pHS-cotG-RsNit as a template, amplification was performed using mutant primers V190L-F and V190L-R to obtain a full-length plasmid PCR product containing the V190L mutation. Then, the full-length plasmid PCR product containing the V190L mutation was treated with the restriction endonuclease Dpn I. After selectively digesting the original template, the DpnI-treated full-length plasmid PCR product containing the V190L mutation was transformed into E. coli DH5α competent cells. Plasmid was extracted to obtain the mutant plasmid DH5α-pHS-cotG-RsNit-V190L.

[0136] The primers for site-directed mutagenesis of the whole plasmid PCR were the two primers of V190L, and their sequences are as follows:

[0137] V190L-F: CTGCTGGGTCCGATTTTTGGTGAACAGATT,

[0138] V190L-R:ACCCAGCAGTGAACCCGGAAAATGGCTC;

[0139] The reaction system for site-directed mutagenesis of whole plasmid PCR is as follows: 1 μL each of 10 μM V190L-F and V190L-R primers, 10 μL of 2×PrimeSTARMax Premix, 1 μL of DH5α-pHS-cotG-RsNit-V190L plasmid template (concentration 0.1-1 ng / μL), and ddH2O to make up to 20 μL.

[0140] The amplification program for whole plasmid PCR was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min 10 s (30 cycles in total); 72℃ final extension for 10 min.

[0141] S2. Using the mutant plasmid DH5α-pHS-cotG-V190L obtained in step S1 as a template, full plasmid PCR was performed using the F194H mutant primers to amplify the PCR product containing the F194H mutation. The PCR product containing the F194H mutation was treated with the restriction endonuclease DpnI to selectively digest the original template plasmid. The DpnI-treated PCR product containing the F194H mutation was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pHS-cotG-RsNit-V190L-F194H, denoted as DH5α-pHS-cotG-RsNit-T1. The nitrile hydrolase mutant gene fragment carried by the mutant plasmid was denoted as RsNit-T1.

[0142] The sequences of the F194H mutation primers (F194H-F and F194H-R) are as follows:

[0143] F194H-F;GGTCCGATTCATGGTGAACAGATTGAAGTT

[0144] F194H-R: CTGTTCACCATGAATCGGACCAACCAGTGA

[0145] The whole plasmid PCR amplification system is as follows: 1 μL of 10 μM F194H-F primer, 1 μL of 10 μM F194H-R primer, 10 μL of 2×PrimeSTARMax Premix, 1 μL of template plasmid DH5α-pHS-cotG-RsNit-V190L (concentration of 0.1-1 ng / μL), and ddH2O to make up to 20 μL.

[0146] Whole plasmid PCR amplification program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min 10 s (30 cycles in total); 72℃ final extension for 10 min.

[0147] (5) The mutant structures were predicted using SWISS-MODEL. The nucleotide sequences of mutant T1 are shown in SEQ ID NO. 5, mutant T2 in SEQ ID NO. 7, mutant T3 in SEQ ID NO. 8, and mutant T4 in SEQ ID NO. 9. Tertiary structure models of the four two-point mutations (T1, T2, T3, and T4) were established. Molecular docking was performed using AutoDockVina software, and the interaction between the protein and the ligand 4-pentenonitrile was visualized using Discovery Studio 2019 software. The results are shown in... Figure 11 As shown. Through force analysis, it was found that mutants T1 and T2 had relatively consistent mutation types, as did mutants T3 and T4. Therefore, mutants T1 and T4 were selected as representatives for molecular dynamics simulation using GROMACS. Root mean square bias (RMSD) is often used to evaluate whether the simulation system has reached stability. Figure 12 The average RMSD values ​​of the RsNit, T1 (V190L / F194H), and T4 (V190T / F194S) protein ligands were 0.2239±0.0456 nm, 0.2129±0.0367 nm, and 0.1864±0.0521 nm, respectively, all reaching equilibrium after 80 ns, indicating that the entire simulation process was stable and reliable. Furthermore, the decrease in RMSD values ​​after mutation suggests greater stability.

[0148] RMSF calculations represent the fluctuations of each atom relative to its average position, characterizing the structural changes over time and providing a representation of the flexibility of different regions of the protein. From the figure, we can see that the average RMSF values ​​for RsNit, T1 (V190L / F194H), and T4 (V190T / F194S) are 0.0940 nm, 0.0760 nm, and 0.0895 nm, respectively. This indicates that the wild-type RsNit residues are more flexible, resulting in weaker binding affinity, while the mutants are more stable in binding to the substrate. T1 exhibits the most stable overall structure, possibly because the mutation introduces hydrophobic residues leucine and histidine, enhancing the stacking effect of the protein core. Studies of key local residues revealed that the RMSF values ​​at the C161 site in mutants T1 and T4 were significantly lower than those in the wild type by 75% and 70%, respectively. This indicates that local rigidification at the C161 site is beneficial for the function of the catalytic triad. Furthermore, the RMSF values ​​of T1 at sites 190 and 194 were significantly lower than those of the wild type, while the RMSF values ​​of T4 at sites 190 and 194 were significantly higher than those of the wild type. This suggests that the effect of mutations on RMSF values ​​is more reflected at the C161 site of the catalytic triad.

[0149] Solvent-accessible surface area (SASA) is calculated by analyzing the solute surface area through van der Waals forces and interactions with solvent molecules. Changes in SASA can predict changes in protein structure. The results show that the T1 and T4 structures significantly reduced the solute surface area compared to the wild type, indicating that mutations may lead to conformational contraction or rearrangement of hydrophobic regions. This conformational contraction also explains why reduced nonspecific solvent interactions can lead to increased thermal stability, which is consistent with the experimental observation of a significant increase in the thermal stability of T1.

[0150] The binding energies of nitrile hydrolases to their substrates are shown in the table. In the enzyme-substrate complex systems, the binding free energies of the histone ligands in the RsNit, T1 (V190L / F194H), and T4 (V190T / F194S) complexes were negative: -15.862 KJ / mol, -17.633 KJ / mol, and -18.022 KJ / mol, respectively. This indicates that the binding abilities of mutants T1 and T4 are superior to those of the wild type. Furthermore, the dominant interaction energy is van der Waals interaction. The results show that mutants T1 and T4 significantly improved the binding affinity between the enzyme and substrate by enhancing van der Waals forces, electrostatic interactions, and nonpolar solvation energy, with T1 being the optimal mutant.

[0151]

[0152] (6) Further enzymatic analysis of mutant T1 was performed, and the results are as follows: Figure 13As shown in the figure. The optimal pH for mutant T1 is 8.0, and it is completely inactivated at pH 9.0. Compared with the wild type, this mutant is more readily inactivated in alkaline environments. Defining enzyme activity at pH 8.0 as 100%, this mutant retains 34% and 28.2% of its activity at pH 5.0 and pH 4.0, respectively, while the wild-type RsNit is completely inactivated at pH 4.0. This indicates that mutant T1 has better enzyme activity in acidic environments than the wild type. The activity at pH 7.0 is 116.95 ± 3.22 U / mL. The optimal temperature for mutant T1 is as follows: Figure 13 As shown in b, the optimal temperature for this mutant is 40℃. It retains over 80% of its enzyme activity at 70℃, 35.8% at 80℃, and nearly 20% at 90℃ and 100℃. In contrast, the wild-type enzyme activity is less than 30% at 70℃ and nearly inactive at 80℃. This indicates that mutant T1 has extremely high heat resistance compared to the wild-type. Further investigation into its pH tolerance was conducted by incubating mutant T1 at pH 4.0 and pH 5.0, with the following results: Figure 13 As shown in c, T1 retained approximately 50% activity after 1 hour of incubation at pH 5.0 and 4.0, and over 20% activity after 3 hours of incubation. In contrast, the wild-type enzyme activity significantly decreased to approximately 20% after 1 hour of incubation at pH 5.0. The mutant T1 exhibited significantly improved acid tolerance compared to the wild-type. The mutant T1 was incubated at 80°C, and the results are as follows... Figure 13 As shown in d, the enzyme activity was about 60% retained after 60 min of incubation and about 50% retained after 5 h of incubation, showing excellent heat resistance compared to the wild type.

[0153] The substrate profile of mutant T1 was determined, and the results are shown in the table. The enzyme activity of mutant T1 increased with increasing substrate carbon chain length, a trend consistent with wild-type RsNit. Furthermore, mutant T1 showed increased activity against the most abundant nitrile in rapeseed meal (3-hydroxypent-4-enyl nitrile). Mutant T1 retained approximately 28.3% of its activity against 3-hydroxypent-4-enyl nitrile, a previously unreported finding for nitrile hydrolases degrading nitrile substances in rapeseed meal. This indicates that mutant T1 exhibits good enzyme activity against aliphatic nitrile substances widely present in rapeseed meal, efficiently degrading 4-pentenyl nitrile, and for the first time demonstrates activity against the most abundant nitrile in rapeseed meal, 3-hydroxypent-4-enyl nitrile, with an activity of 33.1 ± 2.14 U / mL.

[0154]

[0155] Example 3: Application of enzyme preparations in fermented rapeseed meal

[0156] Preparation of enzyme preparation: Using Bacillus subtilis DB403 as the host bacterium, the mutant plasmid DH5α-pHS-cotG-RsNit-T1 was introduced by chemical transformation to obtain the engineered bacterium B. subtilis DB403-pHS-cotG-RsNit-T1. The engineered strain *B. subtilis* DB403-pHS-cotG-RsNit-T1 was inoculated into LB medium at a 1% inoculum. After a first culture (37℃, 180 rpm for 12 h), a culture was obtained. This culture was then inoculated into Difco sporulation medium (DSM) at a 1% inoculum. After a second culture (37℃, 180 rpm for 36 h), the bacterial pellet was collected by centrifugation. The pellet was resuspended in GTE buffer containing lysozyme. The resuspended spores were incubated (37℃, 45 min), centrifuged, and washed with PBS buffer. The pellet was then resuspended in PBS buffer again to obtain a recombinant spore suspension (OD) exhibiting nitrile hydrolase on its surface. 600 If 1), then the mutant enzyme preparation is obtained, denoted as cotG-RsNit-T1.

[0157] The concentration of lysozyme in the GTE Buffer is 2 mg / mL. The components of the GTE Buffer are: 10 mM EDTA, 20 mM Tris-HCl (pH 7.5), and 50 mM Glucose. The GTE buffer consists of: 50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL, and pH 7.5.

[0158] Control: cotG-RsNit spore solution and cotG empty spore solution were prepared in Example 1.

[0159] (1) Extraction of glucosinolates

[0160] Weigh 1g of rapeseed meal and add 20mL of 70% ethanol; extract at 70℃ for 30min, shaking and mixing every ten minutes; filter the supernatant with qualitative filter paper, repeat three times, and finally adjust the volume to 60mL. Perform three parallel trials simultaneously.

[0161] (2) Quantitative analysis of glucosinolates

[0162] A 10 mg / ml stock solution was prepared using potassium glucosinolate as a standard. This stock solution was then diluted to concentrations of 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, and 0.8 mg / ml. 2 mL of glucosinolate solution, 4 mL of sodium carboxymethyl cellulose solution, and 2 mL of palladium chloride solution were added sequentially to 25 mL test tubes. After shaking well and standing for 1 hour, the absorbance was measured at 540 nm. Three replicates were set up for each group; the standard curve for glucosinolates is shown below. Figure 14 As shown.

[0163] The glucosinolate content can be determined by taking 2 mL of the extracted glucosinolate extract and detecting it using the method described above. Take another 2 mL of glucosinolate extract and add 4 mL of sodium carboxymethyl cellulose solution and 2 mL of 0.03 mol / L HCl as one control group; then take 2 mL of pure water and add 4 mL of sodium carboxymethyl cellulose solution and 2 mL of palladium chloride solution as another blank control group.

[0164] (3) To investigate whether nitrile hydrolase preparations have an effect on fermented rapeseed meal, the following six groups were set up: Lactobacillus plantarum was added at 10% of its mass, enzyme preparations were added at 10%, and calcium carbonate (Ca) was added at 10%. 2+ The addition amount is 2%, the total moisture content of the feed is 40%; the fermentation temperature is 25℃, and the fermentation time is 7 days.

[0165] 1. Unfermented group: Only rapeseed meal is mixed with water (unfermented group);

[0166] 2. Rapeseed meal + lactic acid bacteria: Add Lactobacillus plantarum and water to the milled rapeseed meal;

[0167] 3. Rapeseedmeal+lactic acidbacteria+Ca 2+ The milled rapeseed meal was fortified with Lactobacillus plantarum and calcium. 2+ and water;

[0168] 4. Rapeseed meal + lactic acid bacteria tpHS-cotG: Add Lactobacillus plantarum and cotG empty spore liquid and water to the milled rapeseed meal;

[0169] 5. Rapeseedmeal+lactic acidbacteriatpHS-cotG-RsNit+Ca 2+ The milled rapeseed meal was supplemented with Lactobacillus plantarum, cotG-RsNit spore liquid, and Ca. 2+ and water;

[0170] 6. Rapeseedmeal+lactic acidbacteria+pHS-cotG-Tl+Ca 2+ The milled rapeseed meal was fortified with Lactobacillus plantarum, cotG-RsNit-T1 spore enzyme preparation, and Ca. 2+ and water;

[0171] The glucosinolates in each group were quantified after five days of fermentation. Figure 15 As shown, the glucosinolate content in the unfermented group was 31.8 mg / g; the glucosinolate content in the control group fermented with only *Lactobacillus plantarum* was 30.2 mg / g; and the glucosinolate content in the control group fermented with *Lactobacillus plantarum* and Ca... 2+ The control group undergoing fermentation had a glucosinolate content of 31.5 mg / g; *Lactobacillus plantarum*, cotG empty spore liquid, and Ca were added. 2+ The fermentation group had a glucosinolate content of 29.4 mg / g; while the group with added Lactobacillus plantarum, cotG-RsNit spore liquid, and Ca... 2+ The glucosinolate content in the fermented group decreased slightly to 27 mg / g; *Lactobacillus plantarum*, cotG-RsNit-T1 mutant spore liquid, and Ca were added. 2+ The glucosinolate content in the fermentation group decreased significantly, down to 19.2 mg / g, which was about 37% lower than the fermentation group without added nitrile hydrolase. The addition of nitrile hydrolase can promote the positive feedback of glucosinolate degradation into nitrile, thereby indirectly reducing the glucosinolate content.

[0172] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

[0173] sequence list

[0174] SEQ ID NO.1RsNit mature fragment nucleotide sequence

[0175] ATGCCGAAAACAGTGCGTGCGGCAGCGGGTGCAGATTGCACCGGATCTGGCGTCACGTGCAGGTACAGTGGAACGTGTGCTG

[0176] AATGCAATTGCAGAAGCGGCAGATAAAGGTGCAGAACTGATTGTGTTTCCGGAAACCTTTGTGCCGTGGTATCCGTATTTTA

[0177] GCTTTGTTCTGCCGCCGGTTCAGCAGGGCCTGGAACATCTGCGTCTGTATGAAGAAGCGGTTACAGTTCCGAGCCCGGAAA

[0178] CCCATGCAGTTGCAGAAGCAGCGCGCAAACGTGGTGCGGTTGTGGTGCTGGGCGTGAATGAACGCGATCATGGCTCACTGT

[0179] ATAATACACAGCTGATTTTTGATGCGGATGGTACACTGAAACTGAAACGTCGTAAAATTACACCGACATATCATGAACGTATG

[0180] ATTTGGGGTCAGGGCGATGGTGCGGGCCTGAAAGTGGTGGAAACCAAAGTTGGTCGCATGGGTGCACTGGCGTGCTGGGA

[0181] ACATTATAATCCGCTGGCGCGCTATGCGCTGATGGCACAGCATGAAGAAATTCATGCGAGCCATTTTCCGGGTTCACTGGTTG

[0182] GTCCGATTTTTGGTGAACAGATTGAAGTTACAATGCGTCATCATGCACTGGAATCAGGTTGTTTTGTTGTGAATGCAACAGGC

[0183] TGGCTGACCGAAGAACAGATTGCAAGCATTCATCCGGATCCGAAACTGCAGAAAGGCCTGCGCGATGGTTGTATGACCTGTA

[0184] TTATTACACCGGAAGGCCGTCATGCAGCACCGCCGCTGACAGCAGGTGAAGGTATGGTGATTGCGGATCTGGATATGGCGCT

[0185] GATTACAAAACGCAAACGCATGATGGATAGCGTTGGCCATTATGCACGTCCGGAACTGCTGCGCCTGGTGCAGGATGCGCGC

[0186] CCTCAGCGTCCGCTGGAACAGATTCTGCCGGAAGCGACCCCGGAAGCAAGCGAAGCACAGGATGCGSEQ ID NO.2:

[0187] MPKTVRAAAVQIAPDLASRAGTVERVLNAIAEAADKGAELIVFPETFVPWYPYFSFVLPPVQQGLEHLRLYEEAVTVPSPETHAV

[0188] AEAARKRGAVVVLGVNERDHGSLYNTQLIFDADGTLKLKRRKITPTYHERMIWGQGDGAGLKVVETKVGRMGALACWEHYN

[0189] PLARYALMAQHEEIHASHFPGSLVGPIFGEQIEVTMRHHALESGCFVVNATGWLTEEQIASIHPDPKLQKGLRDGCMTCIITPEGR

[0190] HAAPPLTAGEGMVIADLDMALITKRKRMMDSVGHYARPELLRLVQDARPQRPLEQILPEATPEASEAQDASEQ ID NO.3:

[0191] RsNit-F(SpeI)5’-GGACTAGTATGCCGAAAACAGTGCGTGC-3’

[0192] SEQ ID NO.4:

[0193] RsNit-R(HindIII)5’-CCCAAGCTTGGGCTTTGTTAGCAGCCGGATC-3’

[0194] SEQ ID NO.5:T1 mature fragment nucleotide sequence

[0195] ATGCCGAAAACAGTGCGTGCGGCAGCGGTGCAGATTGCACCGGATCTGGCGTCACGTGCAGGTACAGTGGAACGTGTGCTG

[0196] AATGCAATTGCAGAAGCGGCAGATAAAGGTGCAGAACTGATTGTGTTTCCGGAAACCTTTGTGCCGTGGTATCCGTATTTTA

[0197] GCTTTGTTCTGCCGCCGGTTCAGCAGGGCCTGGAACATCTGCGTCTGTATGAAGAAGCGGTTACAGTTCCGAGCCCGGAAA

[0198] CCCATGCAGTTGCAGAAGCAGCGCGCAAACGTGGTGCGGTTGTGGTGCTGGGCGTGAATGAACGCGATCATGGCTCACTGT

[0199] ATAATACACAGCTGATTTTTGATGCGGATGGTACACTGAAACTGAAACGTCGTAAAATTACACCGACATATCATGAACGTATG

[0200] ATTTGGGGTCAGGGCGATGGTGCGGGCCTGAAAGTGGTGGAAACCAAAGTTGGTCGCATGGGTGCACTGGCGTGCTGGGA

[0201] ACATTATAATCCGCTGGCGCGCTATGCGCTGATGGCACAGCATGAAGAAATTCATGCGAGCCATTTTCCGGGTTCACTGCTGG

[0202] GTCCGATTCATGGTGAACAGATTGAAGTTACAATGCGTCATCATGCACTGGAATCAGGTTGTTTTGTTGTGAATGCAACAGG

[0203] CTGGCTGACCGAAGAACAGATTGCAAGCATTCATCCGGATCCGAAACTGCAGAAAGGCCTGCGCGATGGTTGTATGACCTG

[0204] TATTATTACACCGGAAGGCCGTCATGCAGCACCGCCGCTGACAGCAGGTGAAGGTATGGTGATTGCGGATCTGGATATGGCG

[0205] CTGATTACAAAACGCAAACGCATGATGGATAGCGTTGGCCATTATGCACGTCCGGAACTGCTGCGCCTGGTGCAGGATGCGC

[0206] GCCCTCAGCGTCCGCTGGAACAGATTCTGCCGGAAGCGACCCCGGAAGCAAGCGAAGCACAGGATGCG SEQ ID NO.6: Amino acid sequence of the mature fragment of T1

[0207] MPKTVRAAAVQIAPDLASRAGTVERVLNAIAEAADKGAELIVFPETFVPWYPYFSFVLPPVQQGLEHLRLYEEAVTVPSPETHAV

[0208] AEAARKRGAVVVLGVNERDHGSLYNTQLIFDADGTLKLKRRKITPTYHERMIWGQGDGAGLKVVETKVGRMGALACWEHYN

[0209] PLARYALMAQHEEIHASHFPGSLLGPIHGEQIEVTMRHHALESGCFVVNATGWLTEEQIASIHPDPKLQKGLRDGCMTCIITPEGR

[0210] HAAPPLTAGEGMVIADLDMALITKRKRMMDSVGHYARPELLRLVQDARPQRPLEQILPEATPEASEAQDA SEQ ID NO.7: Nucleotide sequence of the mature fragment of T2

[0211] ATGCCGAAAACAGTGCGTGCGGCAGCGGTGCAGATTGCACCGGATCTGGCGTCACGTGCAGGTACAGTGGAACGTGTGCTG

[0212] AATGCAATTGCAGAAGCGGCAGATAAAGGTGCAGAACTGATTGTGTTTCCGGAAACCTTTGTGCCGTGGTATCCGTATTTTA

[0213] GCTTTGTTCTGCCGCCGGTTCAGCAGGGCCTGGAACATCTGCGTCTGTATGAAGAAGCGGTTACAGTTCCGAGCCCGGAAA

[0214] CCCATGCAGTTGCAGAAGCAGCGCGCAAACGTGGTGCGGTTGTGGTGCTGGGCGTGAATGAACGCGATCATGGCTCACTGT

[0215] ATAATACACAGCTGATTTTTGATGCGGATGGTACACTGAAACTGAAACGTCGTAAAATTACACCGACATATCATGAACGTATG

[0216] ATTTGGGGTCAGGGCGATGGTGCGGGCCTGAAAGTGGTGGAAACCAAAGTTGGTCGCATGGGTGCACTGGCGTGCTGGGA

[0217] ACATTATAATCCGCTGGCGCGCTATGCGCTGATGGCACAGCATGAAGAAATTCATGCGAGCCATTTTCCGGGTTCACTGTCGG

[0218] GTCCGATTTATGGTGAACAGATTGAAGTTACAATGCGTCATCATGCACTGGAATCAGGTTGTTTTGTTGTGAATGCAACAGGC

[0219] TGGCTGACCGAAGAACAGATTGCAAGCATTCATCCGGATCCGAAACTGCAGAAAGGCCTGCGCGATGGTTGTATGACCTGTA

[0220] TTATTACACCGGAAGGCCGTCATGCAGCACCGCCGCTGACAGCAGGTGAAGGTATGGTGATTGCGGATCTGGATATGGCGCT

[0221] GATTACAAAACGCAAACGCATGATGGATAGCGTTGGCCATTATGCACGTCCGGAACTGCTGCGCCTGGTGCAGGATGCGCGC

[0222] CCTCAGCGTCCGCTGGAACAGATTCTGCCGGAAGCGACCCCGGAAGCAAGCGAAGCACAGGATGCGSEQ ID NO.8: Nucleotide sequence of the mature fragment of T3

[0223] ATGCCGAAAACAGTGCGTGCGGCAGCGGTGCAGATTGCACCGGATCTGGCGTCACGTGCAGGTACAGTGGAACGTGTGCTG

[0224] AATGCAATTGCAGAAGCGGCAGATAAAGGTGCAGAACTGATTGTGTTTCCGGAAACCTTTGTGCCGTGGTATCCGTATTTTA

[0225] GCTTTGTTCTGCCGCCGGTTCAGCAGGGCCTGGAACATCTGCGTCTGTATGAAGAAGCGGTTACAGTTCCGAGCCCGGAAA

[0226] CCCATGCAGTTGCAGAAGCAGCGCGCAAACGTGGTGCGGTTGTGGTGCTGGGCGTGAATGAACGCGATCATGGCTCACTGT

[0227] ATAATACACAGCTGATTTTTGATGCGGATGGTACACTGAAACTGAAACGTCGTAAAATTACACCGACATATCATGAACGTATG

[0228] ATTTGGGGTCAGGGCGATGGTGCGGGCCTGAAAGTGGTGGAAACCAAAGTTGGTCGCATGGGTGCACTGGCGTGCTGGGA

[0229] ACATTATAATCCGCTGGCGCGCTATGCGCTGATGGCACAGCATGAAGAAATTCATGCGAGCCATTTTCCGGGTTCACTGCGGG

[0230] GTCCGATTACTGGTGAACAGATTGAAGTTACAATGCGTCATCATGCACTGGAATCAGGTTGTTTTGTTGTGAATGCAACAGG

[0231] CTGGCTGACCGAAGAACAGATTGCAAGCATTCATCCGGATCCGAAACTGCAGAAAGGCCTGCGCGATGGTTGTATGACCTG

[0232] TATTATTACACCGGAAGGCCGTCATGCAGCACCGCCGCTGACAGCAGGTGAAGGTATGGTGATTGCGGATCTGGATATGGCG

[0233] CTGATTACAAAACGCAAACGCATGATGGATAGCGTTGGCCATTATGCACGTCCGGAACTGCTGCGCCTGGTGCAGGATGCGC

[0234] GCCCTCAGCGTCCGCTGGAACAGATTCTGCCGGAAGCGACCCCGGAAGCAAGCGAAGCACAGGATGCGSEQ ID NO.9:T4 mature fragment nucleotide sequence

[0235] ATGCCGAAAACAGTGCGTGCGGCAGCGGTGCAGATTGCACCGGATCTGGCGTCACGTGCAGGTACAGTGGAACGTGTGCTG

[0236] AATGCAATTGCAGAAGCGGCAGATAAAGGTGCAGAACTGATTGTGTTTCCGGAAACCTTTGTGCCGTGGTATCCGTATTTTA

[0237] GCTTTGTTCTGCCGCCGGTTCAGCAGGGCCTGGAACATCTGCGTCTGTATGAAGAAGCGGTTACAGTTCCGAGCCCGGAAA

[0238] CCCATGCAGTTGCAGAAGCAGCGCGCAAACGTGGTGCGGTTGTGGTGCTGGGCGTGAATGAACGCGATCATGGCTCACTGT

[0239] ATAATACACAGCTGATTTTTGATGCGGATGGTACACTGAAACTGAAACGTCGTAAAATTACACCGACATATCATGAACGTATG

[0240] ATTTGGGGTCAGGGCGATGGTGCGGGCCTGAAAGTGGTGGAAACCAAAGTTGGTCGCATGGGTGCACTGGCGTGCTGGGA

[0241] ACATTATAATCCGCTGGCGCGCTATGCGCTGATGGCACAGCATGAAGAAATTCATGCGAGCCATTTTCCGGGTTCACTGACGG

[0242] GTCCGATTTCTGGTGAACAGATTGAAGTTACAATGCGTCATCATGCACTGGAATCAGGTTGTTTTGTTGTGAATGCAACAGG

[0243] CTGGCTGACCGAAGAACAGATTGCAAGCATTCATCCGGATCCGAAACTGCAGAAAGGCCTGCGCGATGGTTGTATGACCTG

[0244] TATTATTACACCGGAAGGCCGTCATGCAGCACCGCCGCTGACAGCAGGTGAAGGTATGGTGATTGCGGATCTGGATATGGCG

[0245] CTGATTACAAAACGCAAACGCATGATGGATAGCGTTGGCCATTATGCACGTCCGGAACTGCTGCGCCTGGTGCAGGATGCGCGCCCTCAGCGTCCGCTGGAACAGATTCTGCCGGAAGCGACCCCGGAAGCAAGCGAAGCACAGGATGCG。

Claims

1. An acid- and heat-resistant nitrile hydrolase mutant, characterized in that, The nitrile hydrolase mutant was obtained by mutating valine (Val) at position 190 to leucine (Leu) and phenylalanine (Phe) at position 194 to histidine (His) in the nitrile hydrolase RsNit, whose amino acid sequence is shown in SEQ ID NO.2; wherein the nucleotide sequence of the nitrile hydrolase RsNit is shown in SEQ ID NO.

1.

2. The acid- and heat-resistant nitrile hydrolase mutant according to claim 1, characterized in that, The amino acid sequence of the acid- and heat-resistant nitrile hydrolase mutant is shown in SEQ ID NO.

6.

3. A gene encoding the acid- and heat-resistant nitrile hydrolase mutant, characterized in that, The nucleotide sequence of the acid- and heat-resistant nitrile hydrolase mutant gene is shown in SEQ ID NO.

5.

4. An expression carrier, characterized in that, The expression vector carries the acid- and heat-resistant nitrile hydrolase mutant gene as described in claim 3.

5. An engineered bacterium expressing an acid- and heat-resistant nitrile hydrolase mutant, characterized in that, The nitrile hydrolase mutant engineered bacteria are obtained by transforming the expression vector described in claim 4 into host cells.

6. The engineered bacterium expressing acid- and heat-resistant nitrile hydrolase mutants according to claim 5, characterized in that, The steps for obtaining the nitrile hydrolase mutant engineered bacteria are as follows: using Bacillus subtilis DB403 as the host bacteria, the mutant plasmid is introduced through chemical transformation to construct the nitrile hydrolase mutant engineered bacteria, denoted as B.subtilis DB403-pHS-cotG-RsNit-T1.

7. A method for preparing an acid- and heat-resistant nitrile hydrolase mutant enzyme preparation, characterized in that, Based on the recombinant engineered bacterium B. subtilis DB403-pHS-cotG-RsNit-T1 described in claim 5 or 6, it is inoculated into DSM medium and cultured. After culture, the recombinant spore suspension with nitrile hydrolase on the surface is obtained by centrifugation; or the spore suspension is freeze-dried to prepare a solid powder. The recombinant spore suspension or solid powder is a mutant enzyme preparation, denoted as cotG-RsNit-T1.

8. The method for preparing the acid-resistant and heat-resistant nitrile hydrolase mutant enzyme preparation according to claim 7, characterized in that, The engineered bacterium *B. subtilis* DB403-pHS-cotG-RsNit-T1 was inoculated into LB medium and cultured for the first time. The culture was then inoculated into DSM medium and cultured for the second time. The bacterial pellet was collected by centrifugation and resuspended in GTE buffer containing lysozyme. The resuspended pellet was incubated, centrifuged again, and washed with PBS buffer. The pellet was then resuspended in PBS buffer to obtain a recombinant spore suspension exhibiting nitrile hydrolase on its surface. Alternatively, the recombinant spore suspension could be freeze-dried to obtain a solid powder. Both the recombinant spore suspension and the solid powder were mutant enzyme preparations, denoted as cotG-RsNit-T1.

9. The method for preparing the acid-resistant and heat-resistant nitrile hydrolase mutant enzyme preparation according to claim 8, characterized in that, The concentration of lysozyme in the GTE Buffer is 2 mg / mL, and the components of the GTE Buffer are: 10 mM EDTA, 20 mM Tris-HCl at pH 7.5, and 50 mM Glucose. The first culture conditions were 37℃, 180 rpm for 12 h; the second culture conditions were 37℃, 180 rpm for 36 h; the GTE buffer composition was: 50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL, pH 7.5; the incubation conditions were: 37℃ for 45 min; the OD of the recombinant spore protein suspension was... 600 The value is 1.

10. The use of the mutant enzyme preparation prepared according to any one of claims 7-9 for the degradation of glucosinolates in fermented rapeseed meal, characterized in that, The steps are as follows: First, inoculate the rapeseed meal with lactic acid bacteria, and then add Ca. 2+ A fermentation system was obtained by combining mutant enzyme preparations, and the water content was controlled during fermentation to achieve the application of degrading glucosinolates in fermented rapeseed meal.

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