Efficient conversion method of nitrile compound
By performing site-directed mutagenesis on nitrile hydratase, a highly active and stable nitrile hydratase mutant was obtained, solving the problems of low catalytic activity and low microbial degradation efficiency of natural nitrile hydratase, and realizing efficient detoxification and resource utilization of industrial wastewater and castor bean cake.
Patent Information
- Application Number
- CN202511487812.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-17
- Publication Date
- 2026-01-09
AI Technical Summary
Existing natural nitrile hydratases have low catalytic activity, are unstable, and are difficult to treat wastewater with complex components, which limits their large-scale application. Microbial degradation methods are inefficient, have long cycles, and poor environmental adaptability, making it difficult to achieve detoxification of industrial cyanide-containing wastewater and castor bean cake.
By performing site-directed mutagenesis on the nitrile hydratase of Pseudomonas nitriliphila, a highly active and stable nitrile hydratase mutant was obtained, and a recombinant genetically engineered bacterium was constructed for the catalytic degradation of toxic nitrile compounds.
It achieves efficient conversion of nitrile pollutants, significantly improves the degradation rate of ricinole and 2-cyanobiphenyl, maximizes the retention of nutrients in castor meal, and provides a green and uniform enzymatic solution.
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Figure CN121294415A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering, specifically relating to a nitrile hydratase mutant, a mutant encoding gene, a recombinant vector containing the mutant encoding gene, a recombinant genetically engineered bacterium containing the mutant encoding gene, and the application of a nitrile hydratase mutant in the catalytic degradation of toxic nitrile compounds. Background Technology
[0002] In nature, nitrile compounds, as a class of organic molecules widely distributed in plants and microorganisms, play a crucial physiological role in their life activities. In plants, for example, 3-indoleacetonitrile can serve as a precursor for the synthesis of auxins, directly participating in their growth regulation processes. Meanwhile, cyanogenic glycosides contained in plants such as Bupleurum and Rhodiola possess ecological defense functions; when subjected to external mechanical damage or biological ingestion, they can release hydrogen cyanide through enzymatic decomposition, effectively deterring pests. Microorganisms mainly convert aldoximes into corresponding nitrile products through metabolic pathways such as aldoxime dehydrogenase. These nitrile products further serve as important intermediates in the synthesis of various amide and carboxylic acid metabolites required by cells. However, despite the aforementioned biological significance of nitrile compounds, their generally strong biotoxicity and potential teratogenic effects cannot be ignored. If released into the environment during industrial production, they may pose a persistent threat to ecological security and public health. To address this issue, utilizing microorganisms and their produced nitrile hydrolases and nitrile hydratases for bioremediation of contaminated areas has become a green and sustainable remediation approach. The core of this strategy lies in converting toxic nitrile compounds into less toxic or readily available amide or carboxylic acid products through enzyme catalysis, thereby achieving efficient removal of pollutants from the environment and gradual restoration of ecological functions.
[0003] Nitrile hydratase (NHase), a key metal-dependent microbial enzyme, can efficiently catalyze the hydration of nitrile compounds to produce corresponding amide products under mild reaction conditions such as ambient temperature and near-neutral pH. Since its discovery, it has been identified and functionally analyzed in various microbial systems. Currently, the biotransformation of nitrile substances by microorganisms mainly follows two enzymatic pathways: one is a cascade reaction involving both nitrile hydratase and amidase, where nitrile is first converted to amide by NHase, and then further hydrolyzed into carboxylic acid and ammonia by amidase; the other pathway involves the direct hydrolysis of nitrile to carboxylic acid by nitrile hydrolase. Although these two pathways are widely distributed in nature, and some nitrile hydrolases have been reported to have the ability to generate amides, NHase shows more significant application advantages in industrial biocatalysis and the remediation of toxic nitrile pollution due to its ability to efficiently accumulate amide products during the reaction and its unique catalytic specificity for cyanide. However, naturally derived nitrile hydratases still face bottlenecks in practical environmental remediation applications, such as limited enzyme activity, poor stability, and weak adaptability to complex pollutant systems. Therefore, the directed evolution of NHase through molecular modification technology to obtain mutant enzymes with higher catalytic efficiency, stronger stability, and wider substrate adaptability has become the core development direction for promoting the large-scale application of this enzyme in the fields of cyanide-containing wastewater purification and agricultural by-product detoxification.
[0004] Cyanide (CN⁻), a highly toxic environmental pollutant, is commonly found in wastewater from industries such as electroplating, metallurgy, and chemicals. Even trace amounts can strongly inhibit cellular respiration, posing a serious threat to aquatic ecosystems and human health. Currently, the treatment of cyanide-containing wastewater mainly relies on physicochemical and biological methods. Although physicochemical technologies such as alkaline chlorination and ozone oxidation are widely used, they generally face problems such as high cost, complex operation, easy generation of secondary pollutants, and accelerated equipment corrosion. In contrast, biodegradation methods, with their advantages of being environmentally friendly, having low operating costs, high specificity, and not generating secondary pollution, show significant application potential. As shown in the chemical formula below, the core of this technology lies in utilizing microbial enzyme systems such as nitrile hydratase (EC4.2.1.84) to efficiently hydrate cyanide groups into low-toxicity products containing formamide groups under ambient temperature and pressure, thereby achieving targeted detoxification. However, natural nitrile hydratase still suffers from limited catalytic activity, insufficient stability, and weak adaptability to complex wastewater components in practical applications, which restricts its large-scale engineering application. Therefore, developing engineered nitrile hydratases with high activity, high stability, and strong environmental adaptability through enzyme molecular modification strategies has become a key direction for advancing the development of biological treatment technologies for cyanide-containing wastewater. On the other hand, castor bean, as one of the world's top ten oilseed crops, holds an important position in the fuel and chemical industries. The castor bean cake produced after oil extraction is rich in protein and minerals, possessing good prospects for resource utilization. However, toxic nitrile compounds such as ricinole (3-cyano-4-methoxy-1-methyl-2-pyridone) contained in castor bean cake are difficult to remove effectively, severely limiting its safe application in feed or protein products. Ricinole is a type of natural piperidine alkaloid with significant toxicity. Because efficient detoxification must be achieved while preserving the nutritional components of the bean cake to ensure its subsequent high-value utilization, developing a biological detoxification method that can selectively degrade ricinole without destroying nutritional components has significant practical implications and promising prospects for widespread application.
[0005] .
[0006] Currently, microorganisms such as *Pseudomonas*, *Bacillus*, and *Nitrobacter* have been proven to have the ability to degrade cyanide and are being tested in biological treatment processes for cyanide-containing wastewater and nitrile-containing toxins such as castor oil cake. For example, studies have shown that a *Pseudomonas* strain screened from soil through enrichment culture can remove more than 70% of ricinoleic acid from castor oil cake. However, these microbial degradation methods generally suffer from common bottlenecks such as long cycles, low efficiency, poor environmental adaptability, and incomplete degradation, making large-scale application difficult in castor oil cake detoxification and industrial cyanide-containing wastewater purification.
[0007] Therefore, it is of research significance and application value to find other efficient microbial enzymes for large-scale application in the fields of cyanide-containing wastewater purification and agricultural by-product detoxification. Summary of the Invention
[0008] This invention aims to overcome the limitations of existing technologies, such as the low catalytic activity and instability of natural nitrile hydratases, which are unable to handle wastewater with complex components, thus restricting their large-scale application. Other microbial degradation methods generally suffer from long cycles, low efficiency, poor environmental adaptability, and incomplete degradation. The invention provides a nitrile hydratase mutant, a mutant encoding gene, a recombinant vector containing the mutant encoding gene, and a recombinant genetically engineered bacterium containing the mutant encoding gene. The invention also applies the nitrile hydratase mutant to the process of catalytically degrading toxic nitrile compounds.
[0009] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A nitrile hydratase mutant derived from an amino acid sequence as shown in SEQ ID NO.2 by site-directed mutation, wherein the mutation site is one or more of the following: (1) position 48 of the β subunit, (2) position 72 of the β subunit, and (3) position 127 of the α subunit.
[0010] As a preferred embodiment, a nitrile hydratase mutant is derived from an amino acid sequence as shown in SEQ ID NO.2 by site-directed mutation, wherein the mutation site is two or more of the following: (1) position 48 of the β subunit, (2) position 72 of the β subunit, and (3) position 127 of the α subunit.
[0011] As a further preferred embodiment, a nitrile hydratase mutant is derived from an amino acid sequence as shown in SEQ ID NO.2 by site-directed mutation, wherein the mutation sites are three of the following: (1) position 48 of the β subunit, (2) position 72 of the β subunit, and (3) position 127 of the α subunit.
[0012] Preferably, the nitrile hydratase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.2 at one or more of the following sites: (1) leucine at position 48 of the β subunit is mutated to phenylalanine; (2) tryptophan at position 72 of the β subunit is mutated to serine; (3) lysine at position 127 of the α subunit is mutated to glutamic acid.
[0013] This invention utilizes rational design and directed evolution strategies to address the challenges posed by the evolutionary processes originating from... Pseudomonas nitriliphilaA highly active and stable broad-spectrum nitrile hydratase mutant was successfully obtained through molecular modification of nitrile hydratase (PnNit). This nitrile hydratase mutant can efficiently catalyze the hydration of various nitrile substances, such as ricinole and 2-cyanobiphenyl contained in industrial wastewater, to generate corresponding non-toxic or low-toxicity amide products. It exhibits excellent tolerance to inhibitors in complex matrices and environmental fluctuations (such as pH and temperature changes). When processing castor bean cake, this enzyme can rapidly and specifically degrade ricinole while maximizing the retention of nutrients such as protein. When treating cyanide-containing wastewater, it can achieve efficient conversion of cyanide to formamide, avoiding secondary pollution. This invention provides a unified, efficient, and green enzymatic solution for the resource utilization of agricultural by-products containing nitrile toxins and the purification of industrial wastewater.
[0014] A nitrile hydratase mutant gene as described above.
[0015] A recombinant vector constructed from the coding gene as described above.
[0016] A recombinant genetically engineered bacterium obtained by transformation of the recombinant vector as described above.
[0017] Preferably, the source is Pseudomonas nitriliphila The amino acid sequence of the nitrile hydratase is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.1.
[0018] Preferably, the amino acid sequence of the nitrile hydratase mutant is one of SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8.
[0019] As a further preferred embodiment, the amino acid sequence of the nitrile hydratase mutant is SEQ ID NO.4.
[0020] Preferably, the nucleotide sequence of the nitrile hydratase mutant is one of SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.7.
[0021] As a further preferred embodiment, the nucleotide sequence of the nitrile hydratase mutant is SEQ ID NO.3.
[0022] The application of the nitrile hydratase mutants described above in the catalytic degradation of toxic nitrile compounds.
[0023] Preferably, the application is as follows: using wet bacterial cells obtained by inducing culture of recombinant genetically engineered bacteria containing nitrile hydratase mutant encoding genes, or crude enzymes or purified enzymes extracted after ultrasonic disruption of wet bacterial cells, as catalysts in the catalytic degradation of ricin alkaloid.
[0024] As a preferred embodiment, the application is as follows: using wet bacterial cells obtained by inducing culture of recombinant genetically engineered bacteria containing nitrile hydratase mutant encoding genes, or crude enzymes or purified enzymes extracted from wet bacterial cells after ultrasonic disruption, as catalysts in feed production.
[0025] This invention uses wet bacterial cells obtained by inducing culture of recombinant genetically engineered bacteria containing a nitrile hydratase mutant encoding gene, or crude enzymes or purified enzymes extracted from wet bacterial cells after ultrasonic disruption, as a catalyst. The wet bacterial cells are first resuspended in pure water and uniformly mixed at a ratio of 0.1 to 1 gram of wet bacterial cells per kilogram of castor bean meal, and then added to castor bean meal. The mixture is reacted for 24 hours at pH 7.4 and 40°C to obtain detoxified castor bean meal for feed processing.
[0026] Preferably, the application is as follows: using wet bacterial cells obtained by inducing culture of recombinant genetically engineered bacteria containing a nitrile hydratase mutant encoding gene, or crude enzymes extracted from wet bacterial cells after ultrasonic disruption, or purified enzymes as catalysts in the catalytic degradation of industrial wastewater containing 2-cyanobiphenyl.
[0027] This invention uses wet bacterial cells obtained by inducing culture of recombinant genetically engineered bacteria containing a nitrile hydratase mutant gene, or crude enzymes or purified enzymes extracted by ultrasonic disruption of wet bacterial cells, as a catalyst. These wet bacterial cells are added to wastewater at a ratio of 0.5 to 5 grams per liter of wastewater. The reaction is carried out at pH 7.4 and 40°C for 12 hours to efficiently degrade 2-cyanobiphenyl in the wastewater, thereby obtaining purified wastewater.
[0028] As a preferred application, the application is as follows: Using wet cells obtained by inducing culture of recombinant genetically engineered bacteria containing a nitrile hydratase mutant gene, or crude enzyme extracted from wet cells by ultrasonic disruption, or purified enzyme as a catalyst, and industrial wastewater containing 2-cyanobiphenyl or castor meal containing ricin as a substrate, the reaction is carried out in a phosphate buffer solution with a pH of 7-8 at 25-50°C. After the reaction is complete, the reaction solution is separated and purified to obtain a non-toxic amide product.
[0029] This invention uses wet bacterial cells obtained by inducing and culturing recombinant genetically engineered bacteria containing a nitrile hydratase mutant gene, or crude enzymes or purified enzymes extracted from wet bacterial cells after ultrasonic disruption, as a catalyst. These are added to cyanide-containing wastewater or castor bean cake containing ricinine to form a reaction system. Preferably, the reaction system uses PB buffer (phosphate buffer) at pH 7.4 as the medium, with a nitrile compound concentration of 0.05–50 mM. The catalytic reaction is carried out at 25–50 °C and 400 rpm, more preferably at 40 °C, thereby efficiently hydrolyzing the nitrile compound into a non-toxic amide product.
[0030] This invention achieves a highly efficient catalytic mutant of nitrile hydratase through rational molecular modification of natural nitrile hydratase, and successfully constructs a recombinant genetically engineered bacterium capable of efficiently expressing this mutant. Further experimental verification shows that the obtained nitrile hydratase mutant exhibits significantly enhanced catalytic activity against various nitrile substrates, increasing the degradation rate of ricinoleic acid in castor bean cake from approximately 50% to over 99%; the degradation rate of 2-cyanobiphenyl, a typical pollutant in industrial wastewater, also reaches over 99%, enabling rapid and near-complete degradation of nitrile pollutants. While efficiently detoxifying ricinoleic acid in castor bean cake, it also maximizes the retention of nutrients such as protein in the castor bean cake, providing a feasible technical approach for the safe and resource-based utilization of castor byproducts and the efficient purification of industrial cyanide-containing wastewater.
[0031] The method for preparing the wet mycelium of the present invention is as follows: Recombinant genetically engineered bacteria containing the gene encoding the nitrile hydratase or nitrile hydratase mutant were streaked onto LB solid medium containing 50 μg / mL kanamycin and incubated overnight at 37°C inverted to activate them and obtain single colonies. Single colonies were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and incubated at 37°C for 24 h. The activated bacterial strain was transferred to fresh LB liquid medium containing 50 μg / mL kanamycin at an inoculum volume of 2%, and cultured at 37°C with shaking at 180 rpm until OD500 was reached. 600 When the concentration reaches 0.6~0.8, add IPTG to a final concentration of 0.1mM for induction, and then continue induction culture at 28℃ and 180rpm for 12h; collect the culture medium and centrifuge at 4℃ and 6000rpm, and the resulting precipitate is the desired wet bacterial cell.
[0032] The method for preparing the crude enzyme solution of the present invention is as follows: The wet bacterial cells were resuspended in PB buffer at pH 7.4 at a dosage of 5 g / L and sonicated for 10 min under ice-water bath conditions (power 400 W, working time 2 seconds, interval 1 second). After the disruption was completed, the bacterial solution was centrifuged and the supernatant was collected to obtain the crude enzyme solution containing nitrile hydratase or nitrile hydratase mutant.
[0033] Therefore, the present invention has the following beneficial effects: This invention, through rational design and molecular modification of nitrile hydratase (PnNit), obtained a series of highly active mutants, among which the nitrile hydratase triple mutant PnNit-L48F / W72S / K127E showed a particularly significant increase in catalytic activity. With 2-cyanobiphenyl as a substrate, its relative enzyme activity was approximately 82 times that of the wild-type nitrile hydratase; with ricinole as a substrate, its relative enzyme activity reached 320 times that of the wild-type nitrile hydratase, and the mutant still maintained its complete catalytic hydrolysis ability. In practical applications, using recombinant E. coli expressing this mutant to treat 0.05 mM 2-cyanobiphenyl resulted in complete substrate conversion within 12 hours at 40°C; treatment of castor bean cake meal at the same temperature for 24 hours reduced the ricinole content from the initial 0.3% to 0.01%–0.02%, demonstrating significant degradation effects. This indicates that the mutant has good application potential in industrial wastewater treatment and feed ingredient detoxification. Attached Figure Description
[0034] Figure 1 This is a diagram showing the optimal temperature for the reaction of castor meal catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E in Example 4 of the present invention.
[0035] Figure 2 The optimal temperature for the reaction of 2-cyanobiphenyl catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E in Example 5 of this invention is shown.
[0036] Figure 3 This is a diagram showing the optimal pH for the reaction of castor meal catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E in Example 6 of the present invention.
[0037] Figure 4 This is the optimal pH diagram for the reaction of 2-cyanobiphenyl catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E in Example 7 of the present invention.
[0038] Figure 5 This is a comparison diagram of the reaction process of nitrile hydratase mutant PnNit-L48F / W72S / K127E and wild-type nitrile hydratase catalyzing the hydrolysis of ricinole in Example 8 of the present invention.
[0039] Figure 6 This is a comparison diagram of the reaction process of the hydrolysis of 2-cyanobiphenyl catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E and the wild-type nitrile hydratase in Example 10 of the present invention. Detailed Implementation
[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.
[0041] Example 1: Site-directed mutagenesis and screening of nitrile hydratase (1) Construction of wild-type engineered nitrile hydratase bacteria Based on the source Pseudomonas nitriliphila The nitrile hydratase (PnNit) was used to construct the pET-28a(+)PnNit plasmid, which was then transformed into the host bacterium E. coli BL21(DE3) to construct the engineered bacterium E. coli BL21(DE3) / pET-28a(+)PnNit. This wild-type nitrile hydratase is designated as nitrile hydratase PnNit, and its nucleotide sequence is shown in SEQ ID NO.1, while its amino acid sequence is shown in SEQ ID NO.2.
[0042] (2) Single point mutation Homology modeling and molecular docking analysis identified three key amino acid sites near the active pocket of nitrile hydratase: LEU 48, TRP 72, and LYS 127. To systematically mutate these sites, saturation mutation primers were designed for each of these sites in the wild-type nitrile hydratase amino acid sequence (see Table 1 below). The recombinant plasmid pET28a(+)-PnNit, containing the wild-type nitrile hydratase gene, was used as a template for full plasmid amplification.
[0043] The PCR reaction system (total volume 50 μL) contains the following components: 25 μL of 2×Phanta Max Buffer, 1 μL of 10 mM dNTP mixture, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of plasmid pET28a(+)-PnNit template, 1 μL of DNA polymerase, and ddH2O to make up to 50 μL.
[0044] The PCR conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 68℃ extension for 3.5 min, for 30 cycles; and a final extension at 68℃ for 10 min.
[0045] After the PCR program is completed, the PCR product is analyzed by 0.9% agarose gel electrophoresis. If the electrophoretic bands are 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℃ for 2 h.
[0046] Table 1: Primer Design for Site-Specific Saturation Mutation .
[0047] (3) Combinatorial mutation Using the plasmid of the mutant E. coli BL21(DE3) / pET-28a(+)-PnNit-L48F (nucleotide sequence shown in SEQ ID NO.3) as a template, the W72S mutation was introduced by amplification of the whole plasmid. The PCR reaction system was consistent with the single mutant construction conditions described above. After verification by agarose gel electrophoresis, the template plasmid was digested with DpnI, and the product was transformed into E. coli BL21(DE3) competent cells and plated on LB plates containing 50 μg / mL kanamycin. The double mutant strain E. coli BL21(DE3) / pET-28a(+)-PnNit-L48F / W72S was obtained by screening. Using this double mutant plasmid as a template, the same whole plasmid amplification method was used to further introduce the K127E site mutation. After PCR, DpnI digestion and transformation, the triple mutant strain E.coli BL21(DE3) / pET-28a(+)-PnNit-L48F / W72S / K127E was finally obtained.
[0048] Example 2: Activity assay of nitrile hydratase mutant for ricinine degradation The reaction system for the activity assay was as follows: 10 mL of PB buffer (pH=7.4) with 5 g / L of wet bacterial cells added. 1 g of castor bean meal was added and mixed thoroughly to form the reaction system. The reaction was initiated at 40°C and 400 rpm. After 24 h of reaction, 1 mL of the reaction solution was taken and 10 μL of 6M hydrochloric acid was added to terminate the reaction. After centrifugation, the ricinine content in the substrate was determined by high-performance liquid chromatography (HPLC).
[0049] The high-performance liquid chromatography (HPLC) detection conditions were as follows: a C18 column (250 mm × 4.6 mm, 5 mm), acetonitrile:water = 1:9 (v / v) as the mobile phase, a flow rate of 0.6 mL / min, an injection volume of 20 μL, a detection wavelength of 308 nm, a column temperature of 40 ℃, and a peak elution time of ricinoleic acid of approximately 12 min.
[0050] Enzyme activity unit (U) definition: The amount of cells required to convert 1 μmol of ricinine per minute under conditions of 40℃ and pH=7.4 is defined as one unit of activity (U). Whole-cell enzyme activities are shown in Table 2 below.
[0051] Table 2: Whole-cell enzyme activity coefficients of ricinine by nitrile hydratase mutants .
[0052] Analysis of the data in Table 2 shows that the catalytic activity of the nitrile hydratase triple mutant PnNit-L48F / W72S / K127E for ricinine is about 320 times higher than that of the wild-type PnNit.
[0053] Example 3: Activity assay of nitrile hydratase mutant for the degradation of 2-cyanobiphenyl The reaction system for the activity assay consisted of 10 mL of PB buffer (pH=7.4) with 0.1 g / L of wet bacterial cells. 0.05 mM 2-cyanobiphenyl was added and mixed thoroughly to form the reaction system. The reaction was initiated at 40°C and 400 rpm. After 12 h of reaction, 1 mL of the reaction solution was taken and 10 μL of 6 M hydrochloric acid was added to terminate the reaction. After centrifugation, the content of 2-cyanobiphenyl in the substrate was determined by high-performance liquid chromatography (HPLC).
[0054] The high-performance liquid chromatography (HPLC) detection conditions were as follows: a C18 column (250 mm × 4.6 mm, 5 mm), acetonitrile:water = 3:2 (v / v) as the mobile phase, a flow rate of 0.6 mL / min, an injection volume of 20 μL, a detection wavelength of 260 nm, a column temperature of 40 ℃, and a peak elution time of approximately 10 min for 2-cyanobiphenyl.
[0055] Enzyme activity unit (U) definition: The amount of cells required to convert 1 μmol of 2-cyanobiphenyl per minute under conditions of 40℃ and pH=7.4 is defined as one activity unit (U). Whole-cell enzyme activities are shown in Table 3 below.
[0056] Table 3: Whole-cell enzyme activity coefficients of nitrile hydratase mutants for 2-cyanobiphenyl .
[0057] Analysis of the data in Table 3 shows that the catalytic activity of the nitrile hydratase triple mutant PnNit-L48F / W72S / K127E for 2-cyanobiphenyl is about 82 times higher than that of the wild-type PnNit.
[0058] Example 4: Determination of the optimal temperature for nitrile hydratase PnNit-L48F / W72S / K127E on the substrate ricin Recombinant genetically engineered bacterial wet cells containing the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E were prepared according to the method in Example 2 within the temperature range of 20~60℃, with a temperature gradient of 5℃. The wet cells were added to 10mL of buffer solution at pH=7.4 at a dosage of 5g / L, with a substrate concentration of 50mM, and reacted at different temperatures for 24h. After the reaction, samples were taken and appropriately diluted, and the catalytic efficiency was analyzed by high-performance liquid chromatography. The optimal temperature for the reaction of castor bean meal catalyzed by the nitrile hydratase mutant PnNitL48F / W72S / K127E in this example is shown in the figure. Figure 1 As shown.
[0059] Depend on Figure 1 Analysis showed that the nitrile hydratase mutant PnNit L48F / W72S / K127E exhibited the highest catalytic activity at 40℃. Subsequently, as the temperature continued to rise, the conversion rate decreased, and the enzyme activity showed a gradual downward trend.
[0060] Example 5: Determination of the optimal temperature for nitrile hydratase PnNit-L48F / W72S / K127E on the substrate 2-cyanobiphenyl A temperature range of 20–60 °C was set, with a temperature gradient of 5 °C. Recombinant genetically engineered bacterial wet cells containing the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E were prepared according to the method in Example 3. The wet cells were added to 10 mL of buffer solution at pH 7.4 at a concentration of 0.1 g / L, with a substrate concentration of 0.05 mM. The reaction was carried out at different temperatures for 12 h. After the reaction, samples were taken, appropriately diluted, and analyzed by high-performance liquid chromatography (HPLC). The optimal temperature for the reaction of 2-cyanobiphenyl catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E in this example is shown in the figure. Figure 2 As shown.
[0061] Depend on Figure 2 Analysis shows that the catalytic efficiency of the nitrile hydratase mutant PnNit-L48F / W72S / K127E is the highest at 40℃, and its activity gradually decreases as the temperature increases thereafter.
[0062] Example 6: Determination of the optimal pH of nitrile hydratase PnNit-L48F / W72S / K127E on the substrate ricin The optimal reaction pH for the nitrile hydratase mutant was investigated by varying the pH of the reaction system using different buffer solutions. The pH range was 5.0–9.0, with a gradient of 0.5. Specifically, pH 5.0–6.5 was achieved using a 100 mM citrate-sodium citrate buffer; pH 6.5–8.0 was achieved using a 100 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (PB buffer); and pH 8.0–9.0 was achieved using a 100 mM Tris-hydrochloric acid buffer. Recombinant genetically engineered bacterial wet cells containing the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E were prepared according to the method described in Example 2. The cells were added to 10 mL of buffer at a concentration of 5 g / L, with a ricinine substrate concentration of 50 mM. The reaction was carried out at 40 °C for 24 h. After the reaction, samples were taken, appropriately diluted, and analyzed by high-performance liquid chromatography (HPLC). In this embodiment, the optimal pH for the reaction of castor meal catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E is shown in the figure below. Figure 3 As shown.
[0063] Depend on Figure 3 Analysis showed that the nitrile hydratase mutant PnNit-L48F / W72S / K127E exhibited the best catalytic efficiency for ricinine at a pH of 7.4.
[0064] Example 7: Determination of the optimal pH of nitrile hydratase PnNit-L48F / W72S / K127E on the substrate 2-cyanobiphenyl The optimal reaction pH for the nitrile hydratase mutant was investigated by varying the pH of the reaction system using different buffer solutions. The pH range was 5.0–9.0, with a gradient of 0.5. Specifically, pH 5.0–6.5 was achieved using a 100 mM citrate-sodium citrate buffer; pH 6.5–8.0 was achieved using a 100 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (PB buffer); and pH 8.0–9.0 was achieved using a 100 mM Tris-hydrochloric acid buffer. Recombinant genetically engineered bacterial wet cells containing the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E were prepared according to the method described in Example 3. These cells were added to 10 mL of buffer at a concentration of 0.1 g / L, with a 2-cyanobiphenyl substrate concentration of 0.05 mM. The reaction was carried out at 40 °C for 12 h. After the reaction, samples were taken, appropriately diluted, and analyzed by high-performance liquid chromatography (HPLC). In this embodiment, the optimal pH for the reaction of 2-cyanobiphenyl catalyzed by the nitrile hydratase mutant PnNit-L48F / W72S / K127E is shown in the figure below. Figure 4 As shown.
[0065] Depend on Figure 4 Analysis shows that the nitrile hydratase mutant PnNit-L48F / W72S / K127E exhibits the best catalytic efficiency for 2-cyanobiphenyl at a pH of 7.4.
[0066] Example 8: Application of recombinant genetically engineered bacteria with optimal nitrile hydratase mutant in the catalytic degradation of ricin. Recombinant genetically engineered bacteria containing the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E were prepared according to the method in Example 2. The wet bacterial cells were resuspended in 10 mL of PB buffer at a concentration of 5 g / L. The concentration of the substrate ricinine was 50 mM. The reaction was carried out at 40°C and pH 7.4 for 24 h. After the reaction, samples were taken, appropriately diluted, and analyzed by high-performance liquid chromatography (HPLC). A comparison of the reaction progress of the nitrile hydratase mutant PnNit-L48F / W72S / K127E and the wild-type nitrile hydratase catalyzing the hydrolysis of ricinine is shown in the figure below. Figure 5 As shown.
[0067] Depend on Figure 5Analysis revealed that the substrate concentration of ricinine in the nitrile hydratase mutant group PnNit-L48F / W72S / K127E was reduced to 3 mM, while the substrate concentration reduction was less pronounced in the wild-type nitrile hydratase group. This indicates that molecular modification enabled the originally low-activity nitrile hydratase to almost completely convert ricinine, demonstrating strong industrial potential.
[0068] Example 9: Application of the optimal nitrile hydratase mutant recombinant genetically engineered bacteria in feed Recombinant genetically engineered bacteria, specifically the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E, were prepared according to the method described in Example 2. The wet bacterial cells were added to 10 mL of pure water at a concentration of 5 g / L and resuspended evenly. 1 g of castor bean meal was then added and mixed to form the reaction system. The reaction was carried out at 40°C and pH 7.4 for 24 h to obtain detoxified castor bean meal. Before the reaction, the ricinine content in the castor bean meal was 0.3%. After 24 h of reaction, liquid chromatography analysis showed that the ricinine content in the castor bean meal had decreased to below 0.01%.
[0069] Example 10: Application of the optimal nitrile hydratase mutant recombinant genetically engineered bacteria in the catalysis of 2-cyanobiphenyl Recombinant genetically engineered bacterial wet cells of the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E were prepared according to the method in Example 3. The wet cells were resuspended in 10 mL of PB buffer at a concentration of 0.1 g / L. The concentration of the substrate 2-cyanobiphenyl was 0.05 mM. The reaction was carried out at 40 °C and pH 7.4 for 12 h. After the reaction, samples were taken, appropriately diluted, and analyzed by high-performance liquid chromatography (HPLC). A comparison of the reaction progress of the nitrile hydratase mutant PnNit-L48F / W72S / K127E and the wild-type nitrile hydratase catalyzing the hydrolysis of 2-cyanobiphenyl is shown in the figure below. Figure 6 As shown.
[0070] Depend on Figure 6 Analysis revealed that the substrate concentration of 2-cyanobiphenyl was reduced to 0.001 mM in the nitrile hydratase mutant group PnNit-L48F / W72S / K127E. The reduction in substrate concentration was less pronounced in the wild-type nitrile hydratase. Through molecular modification, the originally low-activity nitrile hydratase was almost completely converted to 2-cyanobiphenyl, demonstrating strong industrial potential.
[0071] Example 11: Application of the optimal nitrile hydratase mutant recombinant genetically engineered bacteria in the degradation of 2-cyanobiphenyl in industrial wastewater Recombinant genetically engineered bacteria, specifically the optimal nitrile hydratase mutant PnNit-L48F / W72S / K127E, were prepared using the method described in Example 3. The wet bacterial cells were added at a concentration of 0.1 g / L to wastewater containing 0.01% 2-cyanobiphenyl. The reaction was carried out at 40°C and pH 7.4 for 12 hours to obtain detoxified water. Before the reaction, the 2-cyanobiphenyl content in the industrial wastewater was 0.01%. After 12 hours of reaction, liquid chromatography analysis revealed that the 2-cyanobiphenyl content in the industrial wastewater had decreased to below 0.002%.
[0072] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.
Claims
1. A nitrile hydratase mutant, characterized in that, It is derived from the amino acid sequence shown in SEQ ID NO.2 by site-directed mutation, wherein the mutation site is one or more of the following: (1) position 48 of the β subunit, (2) position 72 of the β subunit, (3) position 127 of the α subunit.
2. A nitrile hydratase mutant according to claim 1, characterized in that, It is derived from the amino acid sequence shown in SEQ ID NO.2 by site-directed mutation, wherein the mutation site is two or more of the following: (1) position 48 of the β subunit, (2) position 72 of the β subunit, and (3) position 127 of the α subunit.
3. A nitrile hydratase mutant according to claim 1 or 2, characterized in that, The nitrile hydratase mutant is obtained by mutating one or more of the following sites of the amino acid sequence shown in SEQ ID NO.2: (1) leucine at position 48 of the β subunit is mutated to phenylalanine; (2) tryptophan at position 72 of the β subunit is mutated to serine; (3) lysine at position 127 of the α subunit is mutated to glutamic acid.
4. A gene encoding a nitrile hydratase mutant as described in claim 1.
5. A recombinant vector constructed from the encoding gene of claim 4.
6. A recombinant genetically engineered bacterium obtained by transformation of the recombinant vector according to claim 5.
7. The application of the nitrile hydratase mutant as described in claim 1 or 2 in the catalytic degradation of toxic nitrile compounds.
8. The application according to claim 7, characterized in that, The application is as follows: using wet bacterial cells obtained by inducing culture of recombinant genetically engineered bacteria containing nitrile hydratase mutant encoding genes, or crude enzymes extracted from wet bacterial cells after ultrasonic disruption, or purified enzymes as catalysts in the catalytic degradation of ricin.
9. The application according to claim 7, characterized in that, The application is as follows: using wet bacterial cells obtained by inducing culture of recombinant genetically engineered bacteria containing nitrile hydratase mutant encoding genes, or crude enzymes extracted after ultrasonic disruption of wet bacterial cells, or purified enzymes, as catalysts in the catalytic degradation of industrial wastewater containing 2-cyanobiphenyl.
10. The application according to claim 7, characterized in that, The application is as follows: Using wet cells obtained by inducing culture of recombinant genetically engineered bacteria containing a nitrile hydratase mutant gene, or crude enzymes extracted from wet cells after ultrasonic disruption, or purified enzymes, as catalysts, and industrial wastewater containing 2-cyanobiphenyl or castor meal containing ricin alkaloids as substrates, the reaction is carried out in a phosphate buffer solution with a pH of 7-8 at 25-50°C. After the reaction is complete, the reaction solution is separated and purified to obtain non-toxic amide products.