Nitrilase mutant and application thereof in feed

Abstract

The invention provides a nitrilase mutant and application thereof in feed. Castor cake meal is residues obtained after castor seeds are subjected to oil extraction, contains rich protein and cellulose, and can cause huge harm to livestock due to the fact that the castor cake meal contains highly toxic substances such as ricinine, and application of the castor cake meal in the fields of feed and fertilizer is limited. Therefore, the nitrilase is applied to degradation of ricinine in castor cakes, single-point mutation or combined mutation is carried out on the 165th site, the 190th site and the 221 site of the nitrilase from Streptomyces lunalactis, the specific enzyme activity of the obtained optimal nitrilase mutant is 840 times that of wild type nitrilase, most of ricinine can be hydrolyzed within 24 hours, and the optimal nitrilase mutant can be used for degrading ricinine in castor cakes. The challenge confronted by using the castor cake meal as a feed additive is solved, and the method has a relatively high industrial application value.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to a nitrilase mutant, and application of the nitrilase and the mutant in catalyzing ricinine degradation. Background Art

[0002] Castor is one of the world's top ten oil crops. Castor meal is the residue produced after castor oil is pressed, accounting for about 50%. Castor meal is rich in nutrients, especially protein, which accounts for about 35%. In addition, 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. As one of the countries with the largest area of ​​castor crops in the world, industrial oil pressing will inevitably produce a large amount of castor meal. If these meal can be widely used in the fields of feed and fertilizer, it will produce huge economic benefits.

[0003] Since castor meal contains toxic substances such as ricin (N-methyl-3-cyano-4-methoxy-2-pyridone), ricin and allergens, its application in the feed field is greatly limited. These substances can cause serious damage to poultry and livestock and may even be fatal. Therefore, how to remove toxic substances in the meal is the current main research direction. Ricin and allergens will denature and inactivate at high temperatures and lose their toxicity. Generally, the content is almost zero after the heating and extraction process. Ricin is more stable than ricin, so it is more difficult to remove. Currently, there are physical, chemical and biological methods to remove ricin.

[0004] Although the physical and chemical methods of removing ricinine are simple, the removal process involves high temperature and pressure, the addition of organic reagents and other process operations, which will cause the loss of nutrients in castor cake, and the high energy consumption is not environmentally friendly. The biological method is more economical and safe, and the process conditions are milder. It is mainly through the screening of microorganisms in nature that can degrade and transform ricinine. These microorganisms can use ricinine as a carbon source or nitrogen source in the metabolic process and degrade ricinine during their growth and reproduction. The degradation of ricinine by microorganisms is generally achieved by hydrolyzing cyano groups into carboxyl groups:

[0005]

[0006] Currently known bacteria that can degrade cyanide include Pseudomonas, Bacillus, Nitrobacter, etc. Some researchers added ricinine to the enrichment medium, mixed the soil sample with sterile water and then inoculated it into the medium. After multiple screenings, a strain of Pseudomonas with the ability to degrade ricinine was obtained, which can remove more than 70% of ricinine in castor cake. However, microbial degradation of ricinine usually takes a long time, the degradation efficiency is not high, and the degradation is not thorough. Therefore, it is necessary to develop a more efficient method for biodegrading ricinine. Summary of the invention

[0007] In order to solve the problem of low efficiency and incomplete degradation of ricinine by biological methods in the prior art, the present invention provides a method for degrading ricinine by using nitrilase, mutates the nitrilase, obtains a highly active nitrilase mutant, and uses the genetically engineered bacteria of the mutant as a catalyst for the degradation of ricinine in castor cake, with the catalytic activity being 842 times that of the wild type.

[0008] The technical scheme adopted by the present invention is: application of nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis in catalyzing ricinine degradation, the application comprising: using wet bacteria obtained by induction culture of genetic engineering bacteria containing nitrilase encoding genes or crude nitrilase enzyme liquid extracted after ultrasonic crushing of wet bacteria as catalyst, adding ricinine or castor cake containing ricinine to form a reaction system, and hydrolyzing ricinine into N-methyl-3-carboxyl-4-methoxy-2-pyridone.

[0009] Microbial degradation of ricinine usually takes a long time and is not completely degraded, while direct enzyme-catalyzed degradation saves the time required for microbial growth in the reaction body, which is more efficient and convenient. Therefore, the inventor team tried to use nitrilase to degrade ricinine in castor meal, and through activity screening, found that nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis has ricinine degradation activity. Therefore, the present invention proposes to apply nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis to catalyze ricinine degradation, hydrolyze ricinine cyanide to carboxyl while retaining the nutrients in castor meal, and the reaction conditions are mild.

[0010] In order to further improve the degradation efficiency of ricinine by nitrilase, the present invention establishes a high-throughput screening method, constructs a mutant library, and screens out mutants with significantly improved catalytic activity. Therefore, the present invention also provides a nitrilase mutant, which is obtained by single-point mutation or multi-point combined mutation of amino acid sequence No. 165, 190 and 221 of the nitrilase derived from Streptomyces lunaelactis.

[0011] Preferably, the amino acid sequence of the nitrilase from Streptomyces lunaelactis is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO.1.

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

[0013] Preferably, 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 SEQ ID NO.8.

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

[0015] Preferably, 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 SEQ ID NO.7.

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

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

[0018] The present invention also provides the application of the nitrilase mutant in catalyzing the degradation of ricinine, the application comprising: using wet bacteria obtained by induction culture of genetically engineered bacteria containing nitrilase mutant encoding genes or crude enzyme liquid of nitrilase mutant extracted after ultrasonic crushing of wet bacteria as a catalyst, adding ricinine or castor cake containing ricinine, preferably using pH 7-8 buffer as a reaction medium, forming a reaction system, preferably reacting at 25-50°C, more preferably 35°C, 400-600rpm, and hydrolyzing ricinine into N-methyl-3-carboxyl-4-methoxy-2-pyridone. Wherein, in the reaction system, the concentration of ricinine is 10-100mM, more preferably 50mM; the buffer is a pH 7.0, 100mM PB buffer.

[0019] The present invention also provides the application of the nitrilase mutant in feed, which comprises: using wet bacteria obtained by induction culture of genetically engineered bacteria containing a gene encoding the nitrilase mutant or a crude enzyme liquid of the nitrilase mutant extracted after ultrasonic crushing of the wet bacteria as a catalyst, adding the wet bacteria in an amount of 0.1 to 1 g / kg of castor meal to pure water for uniform re-suspending, adding castor meal, and reacting for 12 to 36 hours preferably under the conditions of pH 7 to 8 and 30 to 40° C. to obtain the detoxicated castor meal.

[0020] Wherein, the wet bacteria can be prepared as follows:

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

[0022] (2) A single colony was inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and cultured at 37°C for 12 h;

[0023] (3) Inoculate the culture medium with a volume concentration of 2% into fresh LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and culture at 37°C and 180 rpm until the OD600 is 0.6-0.8. Add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM and induce the culture at 28°C and 180 rpm for 12 h. Centrifuge the culture medium at 8000 rpm and 4°C to collect the precipitate to obtain wet bacteria.

[0024] The crude enzyme solution can be prepared as follows: resuspend the wet bacteria in a pH 7.0, 100 mM PB buffer at a dosage of 50 g / L, ultrasonically disrupt the bacteria on an ice-water mixture for 10 min (power 400 w, disruption 2 s, interval 1 s), centrifuge the resulting bacterial disruption solution at 8000 rpm, and take the supernatant to obtain a crude enzyme solution containing nitrilase or a nitrilase mutant.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention establishes a high-throughput screening method, firstly screens out nitrilase from Streptomyces lunaelactis or Streptomyces hokutonensis that has ricinine degradation activity, and then further constructs a mutant library of nitrilase from Streptomyces lunaelactis, and screens out nitrilase mutants with significantly improved catalytic activity, among which the combined mutant W165G / S190W / V221Y with the highest activity has an enzyme activity 842 times that of the wild-type nitrilase.

[0027] (2) The present invention uses wet bacteria or crude enzyme liquid obtained by fermentation culture of recombinant genetically engineered bacteria containing a mutant nitrilase gene as a catalyst to degrade ricin in castor cake. After 24 hours, the ricinine content is reduced from 0.2% to 0.01%, and the conversion rate can reach 95%, thereby achieving efficient and thorough degradation of ricinine, which is beneficial to the application of castor cake in the fields of feed and fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the optimum temperature diagram for the reaction of the nitrilase mutant SlNit-W165G / S190W / V221Y in Example 3 of the present invention.

[0029] Figure 2 This is the optimum pH diagram for the reaction of the nitrilase mutant SlNit-W165G / S190W / V221Y in Example 4 of the present invention.

[0030] Figure 3 This is a reaction process diagram of the hydrolysis of ricinine catalyzed by the nitrilase mutant SlNit-W165G / S190W / V221Y in Example 5 of the present invention. DETAILED DESCRIPTION

[0031] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0032] Example 1: Screening of Nitrilases with Ricinine Degradation Activity

[0033] Construction of wild-type nitrilase genetic engineering bacteria: The nitrilase encoding genes from different sources in Table 1 were connected to the NcoI and XhoI restriction sites of plasmid pET-28a, and transferred into E.coli BL21 (DE3) by heat shock method, coated on LB plates containing kanamycin at a final concentration of 50 μg / ml, and cultured at 37°C overnight, and positive transformants were selected for sequencing and identification. After verification, the positive monoclonal clone was inoculated into 10 mL of LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and then cultured at 37°C overnight. Subsequently, the plasmid was extracted and verified, and after verification, the recombinant expression vector was transferred into the E.coli BL21 (DE3) strain to obtain the recombinant genetic engineering bacteria E.coli BL21 (DE3) / pET28a (+) -SlNit. Finally, it was cultured overnight at 37°C on an LB plate containing kanamycin at a final concentration of 50 μg / mL. A single clone was picked and inoculated into 10 mL of LB liquid medium containing kanamycin at a final concentration of 50 μg / mL. After culturing overnight at 37°C, the bacterial solution was mixed with 30% glycerol (v / v=1:1) in a glycerol tube and stored in a -80°C refrigerator.

[0034] Inducible expression of recombinant Escherichia coli containing nitrilase: The wild-type nitrilase genetically engineered bacterial strains were inoculated into LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 12 hours, and an inoculum concentration of 2% by volume was inoculated into fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C and 180 rpm until OD600 was 0.6-0.8, and IPTG was added at a final concentration of 0.1 mM, induced culture was carried out at 28°C and 180 rpm for 12 hours, and the culture solution was centrifuged at 8000 rpm and 4°C to obtain the corresponding wet bacteria.

[0035] Screening of nitrilase with ricinine degradation activity: According to the above steps, 52 wild-type nitrilase from different sources were expressed in E. coli BL21 (DE3) and wet cells were obtained, and the obtained wet cells were added to the reaction system for activity screening. The reaction system for activity screening is: total volume 1mL PB (100mM, pH 7.0) buffer, substrate concentration 50mM, wet cell addition amount 10g / L. The reaction was started at 400rpm in a 35℃ water bath, and 10μL 6M hydrochloric acid was added to the reaction solution after 10min of reaction to terminate the reaction. After centrifugation, the substrate ricinine was detected by high performance liquid chromatography. Activity unit (U) definition: At 35℃, pH 7.0, the amount of cells required to convert 1μmol ricinine per minute is regarded as a unit of activity (U). The activities of 52 nitrilases are shown in Table 1. Only the nitrilases from Streptomyces lunaelactis or Streptomyces hokutonensis have catalytic activity towards ricinine.

[0036] Table 1. Activities of 52 nitrilase enzymes

[0037]

[0038]

[0039] Note: Nd = Not Detected.

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

[0041] 1. Saturation mutation

[0042] By 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 amino acid sequence of the wild-type nitrilase in Example 1 was subjected to saturation mutation, and the primers were designed as shown in Table 2. The recombinant plasmid pET28a(+)-SlNit containing the wild-type nitrilase gene was used as a template to perform full plasmid amplification.

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

[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, and 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 is completed, the PCR product is analyzed by 0.9% agarose gel electrophoresis. If the electrophoresis band is verified to be correct, 20 μL of the PCR product is taken, 1 μL of DpnⅠ is added for demethylation, and the template DNA is removed by enzyme digestion at 37°C for 2 h.

[0046] Table 2. Site-directed saturation mutagenesis primer design table

[0047]

[0048] 2. Transformation of nitrilase mutants

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

[0050] 3. High-throughput screening

[0051] Pick the single colony from the transformation plate in step 2 into a 96-deep-well plate, pre-add 1 mL of LB liquid culture medium containing 50 μg / mL kanamycin to each well, and culture at 37°C for 12 h; take 200 μL of bacterial solution and transfer it to 800 μL of fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin and 0.1 mM IPTG, and culture at 28°C for 12 h; centrifuge the 96-deep-well plate at 4000 rpm, 4°C for 20 min, discard the supernatant, and wash and resuspend the bacteria with 200 μL PB buffer (100 mM, pH 7.0). 10mM substrate ricinine (reaction concentration is 5mM) dissolved in 200μL buffer was added to each well, and the reaction was carried out at 35℃ for 2h; the reaction solution of the 96-well plate was centrifuged at 4000rpm and 4℃ for 10min, 20μL of supernatant was taken to the black ELISA plate, 180μL of color development working solution (0.1g of o-phthalaldehyde, 50μL of mercaptoethanol dissolved in 20mL of anhydrous ethanol, 9ml of the solution was dissolved in 91mL of 100mM, pH 7.0 PB buffer) was added to each well in advance, and the color was developed at room temperature for 30min; then the fluorescence intensity was measured using an ELISA instrument (excitation wavelength 412nm, emission wavelength 467nm), and the mutants with higher fluorescence intensity than the control group were selected as positive bacteria.

[0052] 4. Rescreening of Nitrilase Mutants and Activity Determination

[0053] The positive bacteria obtained in step 3 were cultured according to the culture conditions of Example 1 to obtain wet mutant cells. The wet cells were weighed and added into 100 mM PB buffer solution with pH 7.0 and suspended evenly.

[0054] The reaction system for the activity assay was: total volume 10 mL PB (100 mM, pH 7.0) buffer, 20 mM, wet bacterial addition amount 10 g / L. The reaction was started at 400 rpm in a 35°C water bath, and after 10 min of reaction, 1 mL of the reaction solution was added with 10 μL 6 M hydrochloric acid to terminate the reaction. After centrifugation, the substrate ricinine was detected using high performance liquid chromatography.

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

[0056] Definition of enzyme activity unit (U): The amount of cells required to convert 1 μmol of ricin per minute at 35°C and pH 7.0 is considered as one activity unit (U). DNA sequencing of positive clones with improved activity showed that tryptophan replaced glycine at position 165, 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 obtained by screening in step 4 was subjected to iterative mutation, and the primers for saturation mutation at position 190 are shown in Table 2. Using the plasmid pET28a-SlNit-W165G of the nitrilase mutant as a template, the amino acid at position 190 was subjected to a second round of saturation mutation, and the positive mutant W165G / S190W was obtained by screening according to step 4.

[0059] The double mutant W165G / S190W with the highest activity obtained in the second round of saturation mutation screening was subjected to iterative mutation, and the primers for saturation mutation at position 221 are shown in Table 2. Using the plasmid pET28a-SlNit-W165G / S190W of the nitrilase mutant as a template, the amino acid at position 221 was subjected to a third round of saturation mutation, and the positive mutant W165G / S190W / V221Y was screened according to step 4.

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

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

[0062]

[0063] Example 3: Determination of the Optimal Reaction Temperature of Recombinant Bacteria Containing Nitrilase Mutants

[0064] A temperature range of 20 to 60°C was set, and the temperature gradient was 5°C. Genetically engineered bacteria containing the optimal nitrilase mutant SlNit-W165G / S190W / V221Y were prepared according to the method of Example 1. 10 g / L was added to 10 mL of pH 7.0 buffer, and the substrate concentration was 50 mM. The reaction was carried out at different temperatures for 12 hours, and the samples were appropriately diluted for high performance liquid chromatography analysis. The results are shown in FIG. Figure 1 As shown, when the temperature is 35°C, the mutant has the highest catalytic efficiency, and then the activity gradually decreases with increasing temperature.

[0065] Example 4: Determination of the Optimal Reaction pH of Recombinant Bacteria Containing Nitrilase Mutants

[0066] The optimal reaction pH of the nitrilase mutant was explored by using different buffers to change the pH value of the reaction system. The pH range was 5.0-9.0, and the gradient was 0.5, wherein pH 5.0-6.5 was 100mM citric acid-sodium citrate buffer; pH 6.5-8.0 was disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and pH 8.0-9.0 was Tris-hydrochloric acid buffer. Genetically engineered bacteria wet cells containing the optimal nitrilase mutant SlNit-W165G / S190W / V221Y were prepared according to the method in Example 1, added to 10mL of buffer at a dosage of 10g / L, the substrate concentration was 50mM, reacted at 35°C for 12h, and samples were appropriately diluted for HPLC detection and analysis. The results are as follows Figure 2 As shown, the catalytic efficiency of the mutant was best when the pH was 7.0.

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

[0068] According to the method of Example 1, genetically engineered bacteria wet cells containing wild-type nitrilase from Streptomyces lunaelactis and optimal nitrilase mutant SlNit-W165G / S190W / V221Y were prepared respectively, and added to 10 mL PB buffer (100 mM, pH 7.0) at an addition amount of 10 g / L, the concentration of substrate ricinine was 50 mM, and the reaction was carried out at 35° C. for 24 h. Samples were taken every 4 h and appropriately diluted for high performance liquid chromatography analysis. When the reaction reached 24 h, the concentration of substrate ricinine in the mutant group was reduced to 47 mM, and the conversion rate reached 95% ( Figure 3 ). The substrate concentration of the wild-type nitrilase was almost unchanged by the genetically engineered bacteria. Through the modification, the originally low-activity nitrilase can almost completely convert ricinine, which enhances the industrial application potential of nitrilase in catalyzing the degradation of ricinine.

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

[0070] The genetically engineered wet bacterial cells of the optimal nitrilase mutant SlNit-W165G / S190W / V221Y were prepared according to the method of Example 1, and the wet bacterial cells were added to 40g of pure water at an addition amount of 0.1g / kg of cake and evenly suspended, and 60g of castor cake was added and mixed to form a reaction system, and the reaction was carried out at 35°C and pH was constantly controlled at 7.0 for 24 hours to obtain the detoxified castor cake. The ricinine content of the castor cake before the reaction was 0.2%, and the ricinine content was reduced to 0.01% after 24 hours of sampling liquid phase detection.

[0071] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A nitrilase mutant, characterized in that The nitrilase mutant is obtained by subjecting the nitrilase derived from Streptomyces lunaelactis to single-point mutation or multi-point combined mutation at No. 165, No. 190 and No. 221 of its amino acid sequence.

2. The nitrilase mutant according to claim 1, wherein The amino acid sequence of the nitrilase from Streptomyces lunaelactis is shown in SEQ ID NO.

2.

3. The nitrilase mutant according to claim 2, characterized in that The nitrilase mutant is obtained by mutating the tryptophan at position 165 to glycine, and / or the serine at position 190 to tryptophan, and / or the valine at position 221 to tyrosine in the amino acid sequence shown in SEQ ID NO.

2.

4. The nitrilase mutant according to claim 1, wherein The amino acid sequence of the nitrilase mutant is one of SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.

8.

5. A gene encoding the nitrilase mutant according to any one of claims 1 to 4.

6. A recombinant vector comprising the gene encoding the nitrilase mutant according to claim 5.

7. A genetically engineered bacterium containing the gene encoding the nitrilase mutant according to claim 5.

8. Use of a nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis in catalyzing the degradation of ricinine, characterized in that: The application comprises: using wet bacteria obtained by induction culture of genetically engineered bacteria containing a nitrilase encoding gene or crude nitrilase enzyme solution extracted after ultrasonic crushing of the wet bacteria as a catalyst, adding ricinine or castor cake containing ricinine to form a reaction system, and hydrolyzing ricinine into N-methyl-3-carboxyl-4-methoxy-2-pyridone.

9. Use of the nitrilase mutant according to any one of claims 1 to 4 in catalyzing the degradation of ricinine, characterized in that: The application comprises: using wet bacteria obtained by induction culture of genetically engineered bacteria containing a nitrilase mutant encoding gene or crude enzyme liquid of the nitrilase mutant extracted after ultrasonic crushing of the wet bacteria as a catalyst, adding ricin or castor cake containing ricin to form a reaction system, and hydrolyzing ricin into N-methyl-3-carboxyl-4-methoxy-2-pyridone.

10. Use of a nitrilase derived from Streptomyces lunaelactis or Streptomyces hokutonensis or a nitrilase mutant according to any one of claims 1 to 4 in feed, characterized in that: The application comprises: using wet bacteria obtained by induction culture of genetically engineered bacteria containing nitrilase or nitrilase mutant coding genes or crude enzyme liquid of nitrilase or nitrilase mutant extracted after ultrasonic crushing of wet bacteria as catalyst, adding the wet bacteria in an amount of 0.1-1 g / kg of castor meal into pure water and resuspending the wet bacteria evenly, adding castor meal into the water, reacting for 12-36 hours under the conditions of pH 7-8 and 30-40° C., and obtaining detoxicated castor meal.

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