Nitrilase mutants and their use in feed

Abstract

The application provides a nitrilase mutant and application thereof in feed. Castor meal is the residue after castor seed oil is pressed, and contains rich protein and cellulose. Because the castor meal contains ricin and other toxic substances, the castor meal can cause great harm to poultry and livestock, and the application of the castor meal in the field of feed and fertilizer is limited. Therefore, the application of nitrilase in degrading ricin in castor meal is provided, and single-point mutation or combined mutation is carried out on the 165th, 190th and 221st sites of nitrilase from Streptomyces lunaelactis. The specific enzyme activity of the optimal nitrilase mutant is 840 times that of wild-type nitrilase, most of the ricin can be hydrolyzed in 24 hours, the challenge of adding castor meal as feed is solved, and the nitrilase mutant has great industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to a nitrilase mutant, and application of the nitrilase and the mutant thereof in catalyzing degradation of gossypol. BACKGROUND

[0002] Castor is one of the world's top ten oil crops, and castor meal is the residue produced after castor seed oil is extracted, accounting for about 50%. Castor meal is rich in nutrients, especially protein, which accounts for about 35%. In addition to protein, these proteins also contain mineral elements such as calcium, phosphorus and potassium. Therefore, castor meal has great application prospects as a fertilizer or feed additive. China is one of the countries with the largest area of castor crop planting in the world, and industrial oil extraction will inevitably produce a large amount of castor meal. If this meal can be widely used in the fields of feed and fertilizer, it will bring huge economic benefits.

[0003] Due to the presence of toxic substances such as gossypol (N-methyl-3-cyano-4-methoxy-2-pyridone), ricin and allergen in castor meal, its application in the feed field has been greatly limited. These substances can cause harm to poultry and livestock, and in severe cases, they can even cause death. Therefore, how to remove toxic substances from the meal is the current main research direction. Ricin and allergen will denature and lose toxicity at high temperatures, so their content is almost zero after the heating extraction process. Gossypol is more stable than ricin, so it is more difficult to remove. Currently, there are physical, chemical and biological methods for removing gossypol.

[0004] Although the physical and chemical methods for removing gossypol are simple, the removal process involves high temperature and high pressure, the addition of organic reagents and other process operations, which can cause the loss of nutrients in the castor meal and consume a lot of energy, and are not environmentally friendly. The biological method is more economical and safe, and the process conditions are milder. It mainly involves screening microorganisms in nature that can degrade and convert gossypol. These microorganisms can utilize gossypol as a carbon source or nitrogen source during the metabolic process, and degrade gossypol during their growth and reproduction. Microorganisms generally degrade gossypol by hydrolyzing the cyano group to a carboxyl group:

[0005]

[0006] Currently known bacteria capable of degrading cyano groups include Pseudomonas, Bacillus, and Nitrobacter. Some researchers have added gossypol to an enrichment medium, mixed soil samples with sterile water, and then inoculated the medium. After several screenings, a Pseudomonas strain with the ability to degrade gossypol was obtained, which can remove more than 70% of gossypol in castor meal. However, microbial degradation of gossypol usually takes a long time, has low degradation efficiency, and is not complete. Therefore, it is necessary to develop a more efficient method for biodegrading gossypol. SUMMARY

[0007] In order to solve the problems of low efficiency and incomplete degradation of ricinine by biological methods in the prior art, the application provides a method for degrading ricinine by using nitrilase, and a high-activity nitrilase mutant is obtained by mutating the nitrilase, and the genetically engineered bacteria of the mutant are used as catalysts for degrading ricinine in castor meal, and the catalytic activity is 842 times that of the wild type.

[0008] The technical solution adopted by the application is: application of nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis in catalyzing degradation of ricinine, which comprises: using wet bacteria or nitrilase crude enzyme solution extracted after ultrasonic disruption of wet bacteria obtained by inducing culture of genetically engineered bacteria containing nitrilase encoding gene as catalyst, adding ricinine or castor meal containing ricinine to form a reaction system, and hydrolyzing ricinine into N-methyl-3-carboxyl-4-methoxyl-2-pyridone.

[0009] Microbial degradation of ricinine usually takes a long time and is not complete, while direct enzymatic degradation saves the time required for microbial growth in the reaction system, and is more efficient and convenient. Therefore, the inventors' team tried to use nitrilase to degrade ricinine in castor meal, and through activity screening, it was found that nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis of Streptomyces genus has ricinine degradation activity. Therefore, the application proposes to apply nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis to catalyze degradation of ricinine, hydrolyze the cyano group of ricinine into carboxyl group while retaining the nutrients in castor meal, and the reaction conditions are mild.

[0010] In order to further improve the degradation efficiency of nitrilase on ricinine, the application establishes a high-throughput screening method, constructs a mutant library, and screens out a mutant with significantly improved catalytic activity. Therefore, the application also provides a nitrilase mutant, which is obtained by single-point mutation or multi-point combined mutation of the 165th, 190th and 221st amino acid sequences of nitrilase derived from Streptomyces lunaelactis.

[0011] As a preferred, the nitrilase derived from Streptomyces lunaelactis has an amino acid sequence as shown in SEQ ID NO. 2 and a nucleotide sequence as shown in SEQ ID NO. 1.

[0012] As a preference, the nitrilase mutant is obtained by mutating the tryptophan at position 165 of the amino acid sequence shown in SEQ ID NO. 2 to glycine, and / or mutating the serine at position 190 to tryptophan, and / or mutating the valine at position 221 to tyrosine; more preferably, the nitrilase mutant is obtained by mutating the tryptophan at position 165 of the amino acid sequence shown in SEQ ID NO. 2 to glycine, and mutating the serine at position 190 to tryptophan, and mutating the valine at position 221 to tyrosine.

[0013] As a preference, the amino acid sequence of the nitrilase mutant is one of SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8, more preferably, the amino acid sequence of the nitrilase mutant is SEQ ID NO. 8.

[0014] The present application also provides a gene encoding the nitrilase mutant.

[0015] As a preference, the nucleotide sequence of the nitrilase mutant is one of SEQ ID NO. 3, SEQ ID NO. 5, and SEQ ID NO. 7, more preferably, the nucleotide sequence of the nitrilase mutant is SEQ ID NO. 7.

[0016] The present application also provides a recombinant vector containing the gene encoding the nitrilase mutant.

[0017] The present application also provides a genetically engineered bacterium containing the gene encoding the nitrilase mutant.

[0018] The present application also provides the use of the nitrilase mutant in catalyzing the degradation of gossypol, which comprises using the wet bacterium obtained by inducing the genetically engineered bacterium containing the gene encoding the nitrilase mutant or the crude enzyme solution of the nitrilase mutant extracted after ultrasonic disruption of the wet bacterium as a catalyst, adding gossypol or gossypol-containing gossypol cake, preferably using a pH 7-8 buffer as a reaction medium to form a reaction system, and preferably performing the reaction at 25-50°C, more preferably at 35°C, at 400-600 rpm, to hydrolyze gossypol into N-methyl-3-carboxyl-4-methoxy-2-pyridone. In the reaction system, the concentration of gossypol is 10-100 mM, more preferably 50 mM; and the buffer is a pH 7.0, 100 mM PB buffer.

[0019] The application further provides the application of the nitrilase mutant in feed, which comprises: using the wet bacteria body obtained by induced culture of the genetically engineered bacteria containing the nitrilase mutant encoding gene or the nitrilase mutant crude enzyme solution extracted after ultrasonic disruption of the wet bacteria body as a catalyst, adding the wet bacteria body into pure water in an adding amount of 0.1-1 g / kg of castor meal to resuspend uniformly, adding the castor meal, preferably reacting for 12-36 h under the condition of pH 7-8 and 30-40 DEG C to obtain the detoxified castor meal.

[0020] The wet bacteria body can be prepared by the following method:

[0021] (1) inoculating the genetically engineered bacteria containing the nitrilase or nitrilase mutant encoding gene into solid LB solid culture medium containing 50 μg / mL kanamycin, activating overnight at 37 DEG C to obtain single colonies;

[0022] (2) inoculating the single colonies into LB liquid culture medium containing 50 μg / mL kanamycin at a final concentration and culturing at 37 DEG C for 12 h;

[0023] (3) inoculating into fresh LB liquid culture medium containing 50 μg / mL kanamycin at a final concentration at a volume concentration of 2% at 37 DEG C and 180 rpm to culture until the OD600 is 0.6-0.8, adding isopropyl-beta-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and then culturing at 28 DEG C and 180 rpm for 12 h, and then centrifuging the culture solution at 8000 rpm and 4 DEG C to collect the precipitate to obtain the wet bacteria body.

[0024] The crude enzyme solution can be prepared by the following method: resuspending the wet bacteria body in pH 7.0, 100 mM PB buffer at a dosage of 50 g / L, ultrasonically disrupting for 10 min on an ice water mixture (the power is 400 w, and the disruption is 2 s with an interval of 1 s), centrifuging the obtained bacteria disruption solution at 8000 rpm, and taking the supernatant to obtain the crude enzyme solution containing the nitrilase or nitrilase mutant.

[0025] The application has the following beneficial effects:

[0026] (1) The present application first screens out nitrilase from Streptomyces lunaelactis or Streptomyces hokutonensis with castorine degradation activity by establishing a high-throughput screening method, and then further constructs a mutant library of nitrilase from Streptomyces lunaelactis, and screens to obtain a nitrilase mutant with significantly improved catalytic activity, wherein the highest activity combination mutant W165G / S190W / V221Y has an enzyme activity of 842 times that of the wild-type nitrilase.

[0027] (2) The wet bacteria or crude enzyme liquid obtained by fermenting and culturing the recombinant genetically engineered bacteria containing the nitrilase mutant gene is used as a catalyst to degrade castorine in castor meal, and after 24 h, the castorine content is reduced from 0.2% to 0.01%, and the conversion rate can reach 95%, realizing efficient and complete degradation of castorine, which is conducive to the application of castor meal in the fields of feed and fertilizer. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a graph of the reaction optimum temperature of the nitrilase mutant SlNit-W165G / S190W / V221Y in Example 3 of the present application.

[0029] Figure 2 is a graph of the reaction optimum pH of the nitrilase mutant SlNit-W165G / S190W / V221Y in Example 4 of the present application.

[0030] Figure 3 is a reaction progress graph of the nitrilase mutant SlNit-W165G / S190W / V221Y catalyzing castorine hydrolysis in Example 5 of the present application. DETAILED DESCRIPTION

[0031] The following specific embodiments illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the present specification. The present application can also be implemented or applied by different specific embodiments, and the details in the specification can be modified or changed based on different views and applications without departing from the spirit of the present application. It should be noted that the following examples and features in the examples can be combined with each other without conflict. In the present application examples, the methods used are conventional methods, and the reagents used can be obtained from commercial channels.

[0032] Example 1: Screening of nitrilase with castorine degradation activity

[0033] Construction of wild-type nitrilase gene engineering bacteria: The nitrilase encoding genes from different sources in Table 1 were ligated to the Ncol and Xhol enzyme digestion sites of plasmid pET-28a, and transformed into E. coli BL21(DE3) by heat shock method, spread on LB plates containing a final concentration of 50 μg / mL kanamycin, and incubated at 37°C overnight, and positive transformants were selected and sequenced. After verification, the positive monoclonal was inoculated into 10 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and then incubated at 37°C overnight. Then, the plasmid was extracted and verified, and the recombinant expression vector was transformed into the E. coli BL21(DE3) strain to obtain the recombinant genetically engineered bacteria E. coli BL21(DE3) / pET28a(+)-SlNit. Finally, the LB plate containing a final concentration of 50 μg / mL kanamycin was incubated at 37°C overnight. The single colony was inoculated into 10 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and incubated at 37°C overnight, then mixed with 30% glycerol (v / v = 1:1) in a glycerol tube, and stored in a -80°C refrigerator.

[0034] Induced expression of recombinant E. coli containing nitrilase: The wild-type nitrilase gene engineering bacteria strains were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and incubated at 37°C for 12 h, and then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 2% inoculum, and incubated at 37°C, 180 rpm until the OD 600 was 0.6-0.8, and then added with a final concentration of 0.1 mM IPTG, and incubated at 28°C, 180 rpm for 12 h, and then centrifuged at 8000 rpm, 4°C to obtain the corresponding wet bacteria.

[0035] Screening of nitrilases with gossypol degradation activity: According to the above steps, 52 different sources of wild-type nitrilases were expressed in E. coli BL21(DE3) and wet bacteria were obtained, and the obtained wet bacteria were added to the reaction system for activity screening. The reaction system for activity screening was: total volume 1 mL PB (100 mM, pH 7.0) buffer, substrate concentration 50 mM, and wet bacteria addition amount 10 g / L. The reaction was started at 400 rpm in a 35°C water bath, and after 10 min, 10 μL 6M hydrochloric acid was added to terminate the reaction, and after centrifugation, high performance liquid chromatography was used to detect the substrate gossypol. The activity unit (U) was defined: the amount of cells required to convert 1 μmol gossypol per minute at 35°C, pH 7.0 was defined as an activity unit (U). The activity of 52 nitrilases is shown in Table 1, only the nitrilases derived from Streptomyces lunaelactis or Streptomyces hokutonensis have catalytic activity for gossypol.

[0036] Table 1. Activity of 52 nitrilase enzymes

[0037]

[0038]

[0039] Note: Nd = Not Detected.

[0040] Example 2: Construction and screening of a nitrilase mutant library

[0041] 1. Saturation mutagenesis

[0042] Through homology modeling and molecular docking, the amino acid sites Trp-165, Ser-190 and Val-221 near the active pocket of nitrilase were screened. The amino acid at position 165 in the wild-type nitrilase amino acid sequence in Example 1 was subjected to saturation mutagenesis, and the primers are shown in Table 2. The recombinant plasmid pET28a(+)-SlNit containing the wild-type nitrilase gene was used as a template for whole plasmid amplification.

[0043] The PCR system (50 uL) was as follows: 2 x phanta Max buffer 25 uL, dNTP mixture (10 mM) 1 uL, forward primer (10 uM) 2 uL, reverse primer (10 uM) 2 uL, plasmid pET28a(+)-SlNit 1 uL, Phanta Max DNA polymerase 1 uL, and ddH2O to 50 uL.

[0044] The PCR conditions were as follows: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 3.5 min, for 30 cycles; and finally extension at 72 °C for 10 min.

[0045] After the PCR program was completed, the PCR product was analyzed by 0.9% agarose gel electrophoresis. If the electrophoresis band was verified to be correct, 20 uL of the PCR product was added with 1 uL of Dpn I demethylation, and the template DNA was removed by enzyme digestion at 37 °C for 2 h.

[0046] Table 2. Design table of site-directed saturation mutagenesis primers

[0047]

[0048] 2. Transformation of nitrilase mutants

[0049] Take competent cells E. coli BL21 (DE3), add 10 μL of PCR product in step 1, stand on ice for 30 min, then heat shock at 42℃ for 90 s, add 600-700 μL of LB liquid medium, cultivate at 37℃ at 180 rpm for 1 h, spread on LB plate containing 50 ug / L kanamycin, cultivate at 37℃ overnight.

[0050] 3. High-throughput screening

[0051] Take single colonies from the transformation plate in step 2 to a 96-deep well plate, previously add 1 mL of LB liquid medium containing 50 μg / mL kanamycin to each well, cultivate at 37℃ for 12 h; take 200 μL of bacterial solution and transfer to 800 μL of fresh LB liquid medium containing 50 μg / mL kanamycin and 0.1 mM IPTG at a final concentration, cultivate at 28℃ for 12 h; centrifuge the 96-deep well plate at 4000 rpm at 4℃ for 20 min, discard the supernatant, wash and resuspend the bacterial cells with 200 μL of PB buffer (100 mM, pH 7.0). Add 10 mM substrate ricin base (reaction concentration 5 mM) dissolved in 200 μL of buffer to each well, react at 35℃ for 2 h; centrifuge the reaction solution of the 96-well plate at 4000 rpm at 4℃ for 10 min, take 20 μL of supernatant to a black enzyme-labeled plate, previously add 180 μL of color developing working solution (0.1 g of o-phthaldehyde, 50 μL of mercaptoethanol dissolved in 20 mL of anhydrous ethanol, take 9 ml of solution and dissolve in 91 mL of PB buffer (100 mM, pH 7.0)), color develop at room temperature for 30 min; then use an enzyme-labeled instrument (excitation wavelength 412 nm, emission wavelength 467 nm) to determine the fluorescence intensity, select mutants with improved fluorescence intensity compared with the control group as positive bacteria.

[0052] 4. Mutant screening and activity determination of nitrile hydratase

[0053] Cultivate the positive bacteria obtained in step 3 according to the cultivation conditions in Example 1 to obtain mutant wet bacterial cells. Weigh the wet bacterial cells and suspend them in 100 mM PB buffer (pH 7.0).

[0054] The reaction system for activity determination is: total volume 10 mL of PB buffer (100 mM, pH 7.0), 20 mM, wet bacterial cell addition amount is 10 g / L. Start the reaction at 400 rpm in a 35℃ water bath, take 1 mL of reaction solution after 10 min of reaction, add 10 μL of 6M hydrochloric acid to terminate the reaction, centrifuge and use high performance liquid chromatography to detect the substrate ricin base.

[0055] The liquid chromatography detection conditions are as follows: the chromatographic column is C18 column (250 mm x 4.6 mm, 5 mm), acetonitrile: water = 1:9 (v / v) is used as the mobile phase, the flow rate is 1 mL / min, the injection amount is 20 uL, the detection wavelength is 308 nm, the column temperature is 40 DEG C, and the ricinine peak time is about 10 min.

[0056] The enzyme activity unit (U) is defined as follows: the amount of cells required for converting 1 μmol of ricinine per minute at 35 DEG C and pH 7.0 is taken as one activity unit (U). The positive clone with improved activity is subjected to DNA sequencing, which shows that the tryptophan at the 165th position is replaced by glycine, and the whole-cell enzyme activity is shown in Table 3.

[0057] 5. Iterative mutation library construction and screening

[0058] The single mutant W165G with the highest activity screened in step 4 is subjected to iterative mutation, and the saturation mutation primer at the 190th position is shown in Table 2. The plasmid pET28a-SlNit-W165G of the nitrilase mutant is taken as the template, the amino acid at the 190th position is subjected to the second round of saturation mutation, and the positive mutant W165G / S190W is screened according to step 4.

[0059] The double mutant W165G / S190W with the highest activity screened in the second round of saturation mutation is subjected to iterative mutation, and the saturation mutation primer at the 221st position is shown in Table 2. The plasmid pET28a-SlNit-W165G / S190W of the nitrilase mutant is taken as the template, the amino acid at the 221st position is subjected to the third round of saturation mutation, and the positive mutant W165G / S190W / V221Y is screened according to step 4.

[0060] The recombinant plasmids pET28a-SlNit-W165G / S190W and pET28a-SlNit-W165G / S190W / V221Y of the positive mutants screened in the iterative mutation are cultured according to the culture conditions in Example 1 to obtain wet bacteria, and the whole-cell enzyme activity is calculated by rescreening and verification according to the method in step 4 (see Table 3).

[0061] Table 3. Whole-cell enzyme activity table of mutants

[0062]

[0063] Example 3: Determination of optimum reaction temperature of nitrilase mutant-containing recombinant bacteria

[0064] A temperature range of 20-60℃ was set, the temperature gradient was 5℃, and the wet bacterial mass of the genetically engineered bacteria containing the optimal nitrilase mutant SlNit-W165G / S190W / V221Y was prepared according to the method of Example 1, 10 mL of buffer solution with pH 7.0 was added at an addition amount of 10 g / L, the substrate concentration was 50 mM, and the reaction was carried out at different temperatures for 12 h. The sample was appropriately diluted for high performance liquid chromatography detection analysis. The results are shown in Figure 1 Figure 6, when the temperature was 35℃, the mutant had the highest catalytic efficiency, and then the activity gradually decreased as the temperature increased.

[0065] Example 4: Determination of the optimal reaction pH of the recombinant bacteria containing the nitrilase mutant

[0066] The optimal reaction pH of the nitrilase mutant was explored by changing the pH value of the reaction system using different buffers. The pH range was 5.0-9.0, and the gradient was 0.5. The pH 5.0-6.5 was 100 mM citric acid-sodium citrate buffer; the pH 6.5-8.0 was sodium phosphate dibasic-sodium phosphate monobasic buffer; and the pH 8.0-9.0 was Tris-hydrochloric acid buffer. The wet bacterial mass of the genetically engineered bacteria containing the optimal nitrilase mutant SlNit-W165G / S190W / V221Y was prepared according to the method of Example 1, 10 mL of buffer solution was added at an addition amount of 10 g / L, the substrate concentration was 50 mM, and the reaction was carried out at 35℃ for 12 h. The sample was appropriately diluted for high performance liquid chromatography detection analysis. The results are shown in Figure 2 Figure 7, when the pH was 7.0, the mutant had the best catalytic efficiency.

[0067] Example 5: Application of the optimal nitrilase mutant genetically engineered bacteria in catalyzing the degradation of gossypol

[0068] The wet bacterial mass of the genetically engineered bacteria containing the wild-type nitrilase from Streptomyces lunaelactis and the optimal nitrilase mutant SlNit-W165G / S190W / V221Y was prepared according to the method of Example 1, 10 mL of PB buffer (100 mM, pH 7.0) was added at an addition amount of 10 g / L, the substrate gossypol concentration was 50 mM, and the reaction was carried out at 35℃ for 24 h. The sample was appropriately diluted for high performance liquid chromatography analysis every 4 h. When the reaction was carried out for 24 h, the substrate gossypol concentration of the mutant group was reduced to 47 mM, and the conversion rate reached 95%( Figure 3 ). The substrate concentration of the wild-type nitrilase genetically engineered bacteria hardly changed. Through modification, the nitrilase with very low activity can approach complete conversion of gossypol, which enhances the industrial application potential of nitrilase in catalyzing the degradation of gossypol.

[0069] Example 6: Application of the optimal nitrilase mutant genetically engineered bacteria in feed

[0070] The genetically engineered bacteria wet bacteria of the nitrilase mutant SlNit-W165G / S190W / V221Y prepared according to the method of Example 1 were resuspended in 40 g of pure water at an addition amount of 0.1 g / kg of cake meal, mixed with 60 g of castor cake meal to form a reaction system, and reacted at 35°C under constant pH control at 7.0 for 24 h to obtain detoxified castor cake meal. The castor base content of the castor cake meal before the reaction was 0.2%, and the castor base content after 24 h was reduced to 0.01% by liquid phase detection.

[0071] The above-described examples only describe the preferred embodiments of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope of the present application.

Claims

1. A nitrile hydrolase mutant, characterized in that, The nitrile hydrolase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.2 to one of the following: (1) tryptophan at position 165 is mutated to glycine, (2) tryptophan at position 165 is mutated to glycine and serine at position 190 is mutated to tryptophan, (3) tryptophan at position 165 is mutated to glycine, serine at position 190 is mutated to tryptophan and valine at position 221 is mutated to tyrosine.

2. The nitrile hydrolase mutant as described in claim 1, characterized in that, The amino acid sequence of the nitrile hydrolase mutant is one of SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.

8.

3. The nitrile hydrolase mutant according to claim 1, characterized in that, The nitrile hydrolase is derived from Streptomyces lunaelactis .

4. A gene encoding a nitrile hydrolase mutant according to any one of claims 1 to 3.

5. A recombinant vector containing the gene encoding the nitrile hydrolase mutant as described in claim 4.

6. Genetically engineered bacteria containing the gene encoding the nitrile hydrolase mutant as described in claim 4.

7. The application of the nitrile hydrolase mutant according to any one of claims 1 to 3 in the catalytic degradation of ricinine, characterized in that, The application includes: using wet bacterial cells obtained by inducing culture of genetically engineered bacteria containing a nitrile hydrolase mutant encoding gene, or crude enzyme solution of nitrile hydrolase mutant extracted by ultrasonic disruption of wet bacterial cells, as a catalyst, adding ricin or castor meal containing ricin to form a reaction system, and hydrolyzing ricin into N-methyl-3-carboxy-4-methoxy-2-pyridone.

8. The application of the nitrile hydrolase mutant according to any one of claims 1 to 3 in feed, characterized in that, The application includes: using wet bacterial cells obtained by inducing culture of genetically engineered bacteria containing a nitrile hydrolase mutant encoding gene, or nitrile hydrolase or crude enzyme solution of nitrile hydrolase mutant extracted by ultrasonic disruption of wet bacterial cells, as a catalyst, adding the wet bacterial cells to pure water at an addition rate of 0.1~1 g / kg castor meal, resuspending them evenly, adding castor meal, and reacting at pH 7~8 and 30~40℃ for 12~36 h to obtain detoxified castor meal.

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