CN118086272A - High-efficiency nitrilase immobilization method and its application - Google Patents

Abstract

The invention discloses a high-efficiency nitrilase enzyme immobilization method and application, wherein the immobilized nitrilase is prepared by adopting an adsorption method or a crosslinking-adsorption method, taking macroporous resin as a carrier, and taking pure enzyme liquid obtained by fermenting and culturing engineering bacteria containing nitrilase encoding genes as an active component; the nucleotide sequence of the nitrilase encoding gene is shown as SEQ ID NO. 1; the total enzyme activity recovery rate of the immobilized nitrilase prepared by the method is more than 77 percent, and the maximum can reach 100 percent. The immobilized nitrilase is recycled, the stability is very good, the nitrilase is transferred to 4 ℃ for 30 days after 30 batches of reaction, 200mM substrate can be completely converted within 2 hours after being taken out, and the conversion rate of each batch of substrate is more than 99%.

Description

High-efficiency nitrilase immobilization method and its application

(I) Technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a method for immobilizing a high-efficiency nitrilase and an application thereof in preparing 1-cyanocyclohexyl acetic acid.

(II) Background technology

Free enzymes are difficult to separate from the reaction and are usually discarded after a single use, which has a high economic cost. Improving the production efficiency of enzymes and extending their service life is a major demand in the field of bioengineering. Enzyme immobilization technology can convert free enzymes into solid state. Immobilized enzymes are more stable and more resistant to environmental changes than free enzymes. Enzymes are easy to separate from the reaction system and have high safety and sustainability. More importantly, immobilized enzymes have strong reusability, which is conducive to continuous operation and rapid termination of reactions, providing more possibilities for the design of bioreactors. Enzyme immobilization technology has been widely used in the pharmaceutical industry, such as as a catalyst to catalyze the synthesis of drugs and intermediates. Immobilized enzymes can also be used to diagnose or treat diseases. Immobilized cells can also be used for coatings of artificial materials to improve biocompatibility. Immobilized enzymes and cells are also widely used in the food industry, nutrition, bioenergy, and textile industries.

Gabapentin, chemical name 1-(aminomethyl)-cyclohexylacetic acid, is an anti-epileptic drug developed by Warner-Lambert and launched in the UK in 1993. Gabapentin has a selective effect on intractable epilepsy (IE), especially partial epileptic seizures (with or without generalized seizures). It has the characteristics of high therapeutic index, good tolerance, low toxicity, no teratogenic or mutagenic effects, short-term and mild side effects, and no serious or chronic adverse reactions. It is a new type of anti-epileptic drug. In recent years, the indications for gabapentin treatment have been continuously expanded, and it is also used to treat neuropathic pain syndromes, including trigeminal neuralgia, perioperative neuralgia, migraine, postherpetic neuralgia, etc. Compared with other similar drugs, gabapentin has better efficacy, fewer toxic side effects, and good tolerance, and is more popular in clinical applications. Relevant data show that the sales market of gabapentin in China has grown rapidly. Gabapentin has been included in the 2020 edition of the Chinese Pharmacopoeia as a Class 1 nervous system drug and a Class 2 anti-epileptic drug. It has also been included in the National Medical Insurance Drug Catalogue and is listed as a Class B drug. In recent years, the amount of gabapentin used in the statistics of terminal anti-epileptic drugs in public medical institutions has continued to rise. In 2019, gabapentin capsules ranked seventh in the generic name ranking of terminal chemical anti-epileptic drugs in China's public medical institutions, accounting for about 4.14% of the market share, but there is still a large gap with the international market. It is predicted that due to the large number of epilepsy and neuralgia patients in China and the world, and the excellent clinical effect of gabapentin, its market still has great development potential, and the global demand for gabapentin drugs and APIs will continue to increase year by year.

There are many synthetic routes for gabapentin, which can be roughly divided into two categories: chemical method and chemical-enzymatic method. Most of the chemical routes that have attracted attention use cyclohexanone as the raw material to chemically synthesize gabapentin through steps such as cyclization, hydrolysis, condensation, and aminolysis: Caiming et al. started from cyclohexanone and obtained the product gabapentin mainly through Wittig reaction, spiro ring construction, rearrangement reaction, ring opening reaction, and ion exchange. Jin-Pei Huang et al. used cyclohexanone to undergo a cyclization reaction under the catalysis of ethanol and monohydrate ammonia. The product was acidified under 80% sulfuric acid to generate dicarboxylic acid, which was condensed with acetic anhydride, and hydroxylamine hydrochloride was added for aminolysis. Finally, it was reacted with sodium perbromate under alkaline conditions of sodium hydroxide to generate the product gabapentin. The product yield of this route is high, but a large amount of concentrated sulfuric acid is required, which produces a large amount of black acidic wastewater, which is highly polluting to the environment; the treatment of this wastewater also requires a large amount of quicklime to neutralize it, which will seriously corrode the equipment and increase the waste residue. In addition, the black product after hydrolysis must be decolorized, which adds another process.

Most chemical synthesis routes of gabapentin have many disadvantages. The chemical-enzymatic synthesis route of using nitrilase to regioselectively hydrolyze the dinitrile substrate to generate a carboxynitrile intermediate, and then reducing the cyano group to generate gabapentin has great advantages such as simple operation, cheap and readily available raw materials, mild reaction conditions, easy control of the reaction process, extremely high catalytic selectivity, high reaction yield, low risk and green environmental protection. Its synthesis route has been verified by the preliminary development of this laboratory and has great potential for industrial application. The chemical-enzymatic synthesis route is as follows (Figure 1); the substrate 1-cyanocyclohexylacetonitrile is catalyzed by nitrilase to generate 1-cyanocyclohexylacetic acid in one step, and then hydrogenated to generate the product gabapentin and its lactam, or another similar route is used, that is, the substrate 1-cyanocyclohexylacetonitrile is catalyzed by nitrile hydratase to generate 1-cyanocyclohexylacetyl, and then hydrolyzed by amidase to generate 1-cyanocyclohexaneacetic acid, and the subsequent steps of generating gabapentin are the same as above.

However, the industrial application of gabapentin chemical-enzymatic method is hindered by poor operation and storage stability of biocatalysts and difficulty in recycling. Free cells or crude enzymes, free enzymes have poor stability as catalysts, are easily denatured and inactivated, and cells or enzymes can only be used once, which increases the cost of enzyme preparations and the discharge of wastewater and waste residues; the mixing of enzymes with substrates and products increases the difficulty of separation and purification of products, affects product yields, and increases production costs. Immobilized enzymes or cells are one of the solutions to these problems. Generally, immobilization can improve the tolerance of enzymes, protect enzymes from adverse conditions, or keep enzyme proteins stable during the reaction process, and realize the recycling of enzymes, reduce the cost of enzyme preparations, and improve product yields.

Immobilized enzyme technology is widely used in biocatalysis. Wu et al. used diatomaceous earth and macroporous resin HPD-417 as adsorption carriers and combined them with cellulose derivative membrane coating to immobilize laccase. The enzyme activity recovery rate of the prepared diatomaceous earth and HPD-417 immobilized laccase can reach 99.33% and 94.15%, and the thermal stability and pH tolerance are greatly improved, which has great potential in environmental governance. Nuraliyah et al. used macroporous anionic resin MP-64 and chitosan-treated macroporous non-ionic resin XAD-7HP to adsorb lipase, and then cross-linked with glutaraldehyde. The enzyme activity recovery rate and specific enzyme activity of the prepared MP-64 resin immobilized enzyme reached 83.79% and 29.41U/g. After the immobilized enzyme was used four times in a cycle, the biodiesel synthesis rate was 70.61% of the initial yield. Meng-Qiu Xu et al. co-immobilized glycerol dehydrogenase and NADH oxidase into cross-linked enzyme aggregates to construct an NAD ⁺ regeneration system. The specific enzyme activity of the cross-linked enzyme aggregate is 950U/g, and its stability to solution pH and temperature is better than that of the free enzyme. After 10 batches of repeated use, the remaining activity is still 63.3%. Its efficiency in catalyzing glycerol to synthesize 1,3-dihydroxyacetone is 2.5 times that of the free enzyme system, and it has good reusability. Lai et al. synthesized amino-functionalized graphene oxide for covalent immobilization of lactase. The immobilization efficiency reached 61% at the optimal temperature, and the enzyme activity recovery was about 95%. Compared with the free enzyme, the immobilized lactase is more stable in acidic and alkaline environments and high temperatures, and can be recycled many times.

It is known from the literature that immobilized enzymes have many advantages and there are many methods for immobilizing enzymes. However, due to the different protein structures of each enzyme, the immobilization methods suitable for the enzyme are also different. It is found from the literature that there are currently few immobilized nitrilase with ideal performance and strong industrialization potential. Therefore, applying immobilized enzyme technology to nitrilase and using it for the synthesis of gabapentin intermediates will have great advantages.

(III) Summary of the invention

The present invention aims to provide an efficient nitrilase immobilization method and its application in preparing 1-cyanocyclohexyl acetic acid, an intermediate of gabapentin. The present invention uses adsorption and/or cross-linking technology to achieve efficient preparation of nitrilase immobilized enzyme, and the obtained immobilized enzyme has high enzyme activity recovery rate, good reusability and excellent storage stability. The obtained immobilized enzyme is applied to catalyze 1-cyanocyclohexyl acetonitrile to synthesize 1-cyanocyclohexyl acetic acid, thereby improving the main indicators such as substrate concentration, conversion rate, enzyme activity recovery rate, reaction batch and the like.

The technical solution adopted by the present invention is:

The invention provides a high-efficiency nitrilase immobilization method for improving enzymatic performance. The immobilization method adopts an adsorption method or a cross-linking-adsorption method, uses a macroporous resin as a carrier, and uses a pure enzyme solution obtained by fermentation and culturing an engineered bacterium containing a nitrilase encoding gene as an active component to prepare immobilized nitrilase; the nucleotide sequence of the nitrilase encoding gene is shown in SEQ ID NO.1, and the amino acid sequence encoding the nitrilase is shown in SEQ ID NO.2.

Furthermore, the pure enzyme solution is prepared as follows: the recombinant Escherichia coli containing the nitrilase encoding gene is inoculated into a test tube of LB liquid culture medium containing a final concentration of 50 μg/mL kanamycin resistance, and cultured at 37°C and 180 rpm for 8 to 12 hours, and then inoculated into a fresh LB liquid culture medium shake flask containing a final concentration of 50 μg/mL kanamycin resistance at an inoculum concentration of 1% by volume, and cultured in a shaker at 37°C and 150 rpm for 2 to 3 hours until the bacterial OD ₆₀₀ reaches 0.6 to 0.8, and then IPTG with a final concentration of 0.1 mM is added, and after induction culture at 28°C for 8 to 12 hours, the supernatant is discarded and the wet bacterial cells are collected; 0.9 g of the wet bacterial cells are taken and suspended in 30 mL The enzyme was ultrasonically disrupted for 30 min in an ice bath at a power of 240 W and a 1-s break and 2-s rest period in a pH 7.0, 200 mM sodium phosphate buffer. The disrupted solution was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was the crude enzyme solution. The crude enzyme solution was purified using a Ni column at a flow rate of 1.5 mL/min. The impurities were eluted with a pH 6-8, 200 mM sodium phosphate buffer containing 50 mM imidazole until the ultraviolet detection signal decreased to the baseline. The Ni column was equilibrated with a pH 6-8, 200 mM sodium phosphate buffer containing 50 mM imidazole, eluted for 5 column volumes, and then eluted with a pH 6-8, 200 mM sodium phosphate buffer containing 50 mM imidazole. 6-8, eluted with 200mM sodium phosphate buffer, collecting the elution peak according to the signal change of the UV detector, dialysis purification of the collected liquid (dialysis bag molecular weight cutoff 14kDa, dialysate using 50mM, pH7 sodium phosphate buffer), taking the intercepted liquid to obtain a pure nitrilase enzyme solution; the sodium phosphate buffer is preferably 200mM, pH 8 sodium phosphate buffer.

Further, the adsorption method is carried out according to the following steps: immersing the macroporous resin in the pure enzyme solution, adsorbing and immobilizing for 1 to 3 hours (preferably 30°C, 130rpm) in a water bath under the conditions of 25 to 55°C and 100 to 200rpm, filtering, washing the filter cake with deionized water for 3 to 5 times, placing it on filter paper and drying it at room temperature, and obtaining immobilized nitrilase, which is recorded as AcN@macroporous resin. The protein concentration of the pure enzyme solution is 0.5-2mg/mL, preferably 1mg/mL; the amount of the pure enzyme solution is 5-15mg/g resin in terms of protein content, preferably 10mg/g resin.

Further, the cross-linking-adsorption method is carried out according to the following steps: the macroporous resin is added to a sodium phosphate buffer (preferably 200 mM, pH 6-8) containing glutaraldehyde at a volume concentration of 0.25-1.00% (preferably 0.5%, v/v), cross-linked for 0.5-3h (preferably 35°C, 130 rpm, 3h) on a shaker at 25-55°C and 100-200 rpm, and washed with deionized water for 3-5 times until glutaraldehyde is no longer detected; then the pure enzyme solution is added, and adsorbed and immobilized for 1-3h (preferably 35°C, 130 rpm, 2h) in a water bath under the conditions of 25-55°C and 100-200 rpm, filtered, and the filter cake is washed with deionized water for 3-5 times, and placed on filter paper to dry at room temperature to obtain immobilized nitrilase, which is recorded as AcN-C@macroporous resin. The volume of the phosphate buffer containing glutaraldehyde is 5-15 mL/g, preferably 10 mL/g, based on the mass of the macroporous resin; the amount of the pure enzyme solution is 5-15 mg/g resin, preferably 10 mg/g resin, based on the protein content.

Furthermore, the macroporous resin model is one of the following: AB-8, 201*7, D001, D113, D115, D201, D730, D750, HPD750, preferably D730 and D750, more preferably D730.

Furthermore, the macroporous resin is pre-activated before immobilization: the macroporous resin is added to an ethanol aqueous solution with a volume concentration of 75-95%, immersed at 25°C for 12 hours, washed with deionized water for 3-5 times until there is no ethanol smell, filtered, and the filter cake is dried in an oven at 65°C to constant weight to obtain the pre-treated macroporous resin; the mass dosage of the macroporous resin is 15-100 g/L based on the volume of the ethanol aqueous solution, preferably 100 g/L.

The present invention also provides an application of the immobilized nitrilase prepared by the method in preparing 1-cyanocyclohexylacetic acid.

The application method comprises the following steps: using immobilized nitrilase as a catalyst, 1-cyanocyclohexylacetonitrile as a substrate, and pH 7.0, 200 mM sodium phosphate buffer as a reaction medium to form an aqueous phase reaction system, reacting for 1 to 3 hours at 25 to 55° C. and 110 to 150 rpm (preferably 35° C. and 130 rpm), and after the reaction is complete, filtering the mixed solution, recovering the immobilized enzyme, and separating and purifying the filtrate to obtain the product 1-cyanocyclohexylacetic acid.

Furthermore, the immobilized nitrilase protein loading is 0.50-10 mg/g (preferably 10 mg/g), the immobilized nitrilase dosage is 20-200 g/L (preferably 100 g/L) based on the volume of the aqueous phase reaction system; and the substrate addition concentration is 0.2-1 M based on the volume of the aqueous phase reaction system.

The filtrate separation and purification method can be a flocculation-extraction method: add polyaluminium chloride with a final concentration of 10 g/L to the reaction solution and stir at room temperature for 4 hours; then add diatomaceous earth with a final concentration of 10 g/L and stir at room temperature for 2 hours; filter to remove solids and collect the filtrate; then use concentrated hydrochloric acid to adjust the pH of the filtrate to 1-3 (preferably 2); add an equal volume of dichloromethane, stir at 350 rpm for 15 minutes, let stand in a separatory funnel for 10 minutes, collect the aqueous phase, and repeat the extraction 3 times; take the organic phase, and rotary evaporate at 25°C until the solid is constant weight to obtain 1-cyanocyclohexylacetic acid.

Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

(1) The high-efficiency nitrile hydrolysis immobilization method provided by the present invention has simple operating conditions, low material cost, and is easy to scale up; the prepared immobilized enzyme has high catalytic efficiency, can be reused in multiple batches, has a long storage time, and is easy to separate.

(2) The total enzyme activity recovery rate of the immobilized nitrilase prepared by the method of the present invention is >77%, and can reach up to 100%. The storage stability of the immobilized nitrilase is very good. After AcN@D730 and AcN-C@D730 were dried in the shade and placed in a refrigerator at 4°C for 6 months, the remaining enzyme activities reached 66.67% and 80.23%, respectively.

(2) 1-Cyanocyclohexylacetic acid (1-CA) was prepared using immobilized nitrilase as a biocatalyst. The biocatalyst concentration was 100 g/L, the substrate concentration was 200 mM, the reaction time was 2 h, the substrate was completely converted, and the product yield was above 98%. The immobilized nitrilase was recycled and had very good reusability and stability. After 30 batches of reactions, it was transferred to 4°C for storage for 30 days. After being taken out, it could still completely convert 200 mM substrate within 2 hours. The substrate conversion rate of each batch was greater than 99%.

(3) The immobilized nitrilase prepared by the method of the present invention requires a small amount of catalyst in the reaction process of preparing 1-cyanocyclohexylacetic acid, has good catalyst reusability and stability, high product yield and purity, and a simple product purification process, and has extremely high application value in the industrial production of gabapentin drug intermediates. The present invention develops a new biocatalytic technology for preparing key gabapentin intermediates, which can simplify the process steps and reduce the discharge of three wastes.

(IV) Description of the drawings

FIG1 is a schematic diagram of the chemical-enzymatic synthesis of gabapentin using 1-cyanocyclohexylacetonitrile (1-CN) as a substrate.

Figure 2 is a map of the expression plasmid pET28b-AcN-T.

(V) Specific implementation methods

The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

The macroporous resins of various types AB-8, 201*7, D001, D113, D115, D201, D730, D750, and HPD750 in the embodiments of the present invention are all purchased from Zhejiang Zhengguang Industrial Co., Ltd.

Preparation of 200 mM sodium phosphate buffer, pH 7.0: weigh 12.17 g of sodium dihydrogen phosphate dihydrate and 43.7 g of disodium hydrogen phosphate dodecahydrate, add deionized water to make up to 1000 mL, and adjust the pH to 7.0.

The Acidovorax facilis ZJB09122 of the present invention is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No.M209044, and has been disclosed in the previously applied patent CN101629192B. The nitrilase of the present invention uses a recombinant nitrilase mutant AcN-T and its encoding gene, which has been disclosed in the previously applied patent CN111172140B.

Example 1: Construction and cultivation of nitrilase genetically engineered bacteria

(1) Construction of nitrilase genetically engineered bacteria:

Referring to patent application CN111172140B, the gene sequence of the mutant AcN-T derived from the nitrilase of A. facilis CCTCCNO:M209044 was synthesized (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2). After sequencing, the amplified fragment was treated with BspHI and BsiWI (GTACC) restriction endonucleases (TaKaRa), and the fragment was connected to the commercial vector pET-28b (Invitrogen) treated with the same restriction endonuclease using T4 DNA ligase (TaKaRa) to construct the expression vector pET-28b (+)-AcN-T (Xiong N, et al. Biochem. Eng. J., 2022, 184: 108475.), and the plasmid map is shown in Figure 2.

The constructed expression vector pET-28b(+)-AcN-T was transformed into Escherichia coli BL21(DE3) (Invitrogen) (42°C, 90s), spread on an LB plate containing 50 μg/mL kanamycin resistance, and cultured at 37°C for 8-12h. Clones were randomly picked and plasmids were extracted for sequencing and identification. Recombinant Escherichia coli BL21(DE3)/pET-28b(+)-AcN-T containing the expression plasmid pET-28b(+)-AcN-T was screened and obtained.

(2) Cultivation of engineered bacteria:

The recombinant Escherichia coli constructed in step (1) was inoculated into a test tube containing LB liquid culture medium with a final concentration of 50 μg/mL kanamycin resistance, and cultured at 37°C and 180 rpm for 8 to 12 h. Then, the inoculum was inoculated into a fresh shake flask containing LB liquid culture medium with a final concentration of 50 μg/mL kanamycin resistance at a volume concentration of 1%, and cultured in a shaker at 37°C and 150 rpm for 2 to 3 h until the bacterial OD600 reached 0.6 to 0.8. IPTG with a final concentration of 0.1 mM was then added, and after induction culture at 28°C for 8 to 12 h, the culture was centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain wet bacterial cells of the recombinant Escherichia coli.

Example 2: Extraction and purification of free nitrilase enzyme

(1) Crude enzyme solution: Weigh 0.9 g of the wet cells prepared in Example 1, suspend in 30 mL of 200 mM, pH 7.0 sodium phosphate buffer, place in ice to maintain a low temperature environment, place in an ultrasonic cell disruptor, adjust the disruption frequency to break for 1 second and stop for 2 seconds, and the disruption power is 240 W. Disrupt for about 30 minutes until the solution is relatively clear. Then place the disrupted solution in a centrifuge tube, centrifuge at 12000 rpm, 4°C for 10 minutes, and collect the supernatant as the crude nitrilase enzyme solution.

(2) Pretreatment of Ni column: Use 5 column volumes of deionized water to wash the Ni column (such as ThermoScientific HisPur Ni-NTA Spin Columns) to remove the ethanol used to preserve the column, then rinse the Ni column with 5 column volumes of pH 8.0 imidazole-free sodium phosphate buffer (containing 50 mM sodium dihydrogen phosphate and 300 mM sodium chloride), and finally rinse with 5 column volumes of pH 8.0, 200 mM sodium phosphate buffer containing 50 mM imidazole until the baseline is stable.

(3) Loading crude protein: The crude enzyme solution was filtered through a 0.45 μm sterile microporous filter membrane and loaded at a flow rate of 1.5 mL/min. The pipeline and Ni column were flushed with a pH 8.0, 200 mM sodium phosphate buffer containing 50 mM imidazole to elute the impurities until the UV detection signal dropped to the baseline and remained stable, which was the Ni column flushing equilibrium.

(4) Elution and sample collection: The Ni column bound to the nitrilase protein after equilibration in step (3) is first rinsed with a pH 8.0, 200 mM sodium phosphate buffer containing 500 mM imidazole. When the signal of the UV detector rises, the elution peak is collected until the UV detector signal drops to the baseline. Collection is stopped to obtain a pure enzyme solution for use after dialysis.

(5) Column regeneration and storage: After eluting the nitrilase, rinse the nickel column with 5 times the column volume of 50 mM imidazole pH 8.0, 200 mM sodium phosphate buffer, then wash the Ni column with 5 times the volume of ultrapure water, and finally rinse with 20% ethanol until the display shows that the conductivity is about 0 mS. Remove the Ni column and store it in a refrigerator at 4°C.

(6) Dialysis purification: First, pre-treat the dialysis bag (molecular weight cutoff 14 kDa) in a boiling water bath for 10 to 15 min, then take it out and cool it. Transfer the pure enzyme solution to the dialysis bag using a pipette. Place the dialysis bag in 50 mM, pH 7 sodium phosphate buffer and dialyze the nitrilase solution containing high imidazole. Clamp both ends of the dialysis bag with leak-proof clamps and place it in a 4°C refrigerator for 12 h. The intercepted liquid is the pure enzyme solution of nitrilase AcN-T.

(7) Gel electrophoresis and protein concentration detection: SDS-PAGE electrophoresis was used to detect the purity of the purified nitrilase protein.

The BCA protein kit was used to measure the absorbance of the bovine serum albumin standard at 562 nm using an enzyme-labeled instrument (the absorbance is proportional to the standard protein content), and finally a real-time standard curve was drawn.

The absorbance of the pure enzyme solution in step (6) at 562 nm was tested using a BCA protein kit, and the protein content in the pure nitrilase solution was calculated based on the standard curve. The protein concentration was adjusted to 1 mg/mL using 200 mM, pH 7 sodium phosphate buffer to obtain the pure nitrilase AcN-T enzyme solution for subsequent experiments.

Example 3: Liquid Phase Detection Method of 1-Cyanocyclohexylacetic Acid (1-CA) and Determination of Nitrilase Activity

1-Cyanocyclohexylacetic acid (1-CA) was analyzed by high performance liquid chromatography. The liquid chromatography instrument was Shimadzu Corporation, the instrument model was SPD-16, and the chromatographic column type was XBridgeBEH C18 Column ( 5μm, 4.6mm×250mm, Waters); mobile phase was 76% buffer (0.58g/L NH ₄ H ₂ PO ₄ and 1.83g/L NaClO ₄ , pH 1.8) and 24% acetonitrile; detection wavelength was 215nm; flow rate was 1.0mL/min; column temperature: 40℃; injection volume: 10μL; analysis time was 20min.

Free enzyme activity assay system: 1 mL (protein concentration 1 mg/mL) of pure enzyme solution of nitrilase AcN-T prepared by the method of Example 2 was taken and kept warm for 10 min at 35° C. and 700 r/min in a water bath, 0.03 g of substrate 1-cyanocyclohexylacetonitrile (1-CN) was added, and the mixture was reacted for 10 min at 35° C. and 700 r/min in a water bath; a trace amount of 6 M hydrochloric acid was added to mix and terminate the reaction, and after centrifugation at 12000 rpm for 1 min, 100 μL of the supernatant was taken and diluted 10 times with 900 μL of deionized water, and the product concentration was detected by liquid chromatography after passing through a 0.22 μm organic membrane, and the enzyme activity was calculated as a free enzyme control.

Resin-immobilized enzyme activity assay system: weigh 1 g (protein loading 10 mg/g) of immobilized nitrilase, add 10 mL of 200 mM, pH 7 sodium phosphate buffer, keep warm at 35°C, 700 r/min in a water bath for 10 min, add 0.3 g of substrate 1-cyanocyclohexylacetonitrile (1-CN), react at 35°C, 700 r/min in a water bath for 10 min; add a trace amount of 6 M hydrochloric acid to mix and terminate the reaction, centrifuge at 12000 rpm for 1 min, take 100 μL of the supernatant, add 900 μL of deionized water to dilute 10 times, pass through a 0.22 μm organic membrane, detect the product concentration by liquid chromatography, and calculate the enzyme activity.

The enzyme activity unit (U) is defined as: at 35°C and 700 r/min, the amount of enzyme required to catalyze the reaction of 1-cyanocyclohexylacetonitrile (1-CN) as a substrate for 10 min, and the amount of enzyme required to catalyze the production of 1 μmol of the product 1-cyanocyclohexylacetic acid (1-CA) per minute is defined as one enzyme activity unit (U).

Example 4: Screening of immobilized carrier resin materials

1g (dry weight) of AB-8, 201*7, D001, D113, D115, D201, D730, D750, and HPD750 resins were accurately weighed respectively, and added to 10mL of 200mM, pH 7.0 sodium phosphate buffer containing 1mg/mL pure enzyme solution of nitrilase AcN-T prepared by the method of Example 2 in terms of protein content, and adsorbed for 5h at 130rpm in a water bath shaker at 35°C, and then filtered by suction. The supernatant was tested for protein content by the method of Example 2, and the protein adsorption amount and protein adsorption rate were calculated, and the adsorption state during the observation process was observed. The obtained filter cake was directly washed with 200mM, pH 7.0 sodium phosphate buffer solution for 3-5 times, and after removing excess water by suction filtration, a resin adsorbing nitrilase was obtained. The adsorption amounts of different macroporous resins are shown in Table 1, and the preferred carrier resins are D730 and D750.

Table 1: Comparison of adsorption capacity of different macroporous resins

Example 5: Two methods for preparing immobilized nitrilase

1. Preparation of immobilized nitrilase by adsorption method:

(1) Resin pretreatment:

Weigh 1 g of macroporous resin D730 and add it into 10 mL of 75% ethanol aqueous solution, soak it at 25°C for 12 h, wash it with deionized water for 3 to 5 times until there is no ethanol smell, filter and dry it, and finally dry it in a 65°C oven for 12 h to constant weight to obtain the pretreated macroporous resin D730; prepare the pretreated macroporous resin D750 by the same method.

(2) Immobilization:

1 g of the macroporous resin pretreated in step (1) was weighed and added to 10 mL of 200 mM, pH 7.0 sodium phosphate buffer containing 1 mg/mL (protein concentration) of pure nitrilase AcN-T prepared by the method of Example 2, and adsorbed for 2 h in a water bath shaker at 35° C., 130 rpm, and the protein adsorption rate was determined by the method of Example 2. The adsorbed macroporous resin was washed with deionized water for 3 to 5 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase by adsorption, which was recorded as AcN@D730 and AcN@D750, and then stored in a refrigerator at 4° C. for future use.

2. Preparation of immobilized nitrilase by cross-linking-adsorption method:

(1) Resin pretreatment:

Weigh 1 g of macroporous resin D730 and add it into 10 mL of 75% ethanol aqueous solution, soak it at 25°C for 12 h, wash it with deionized water for 3 to 5 times until there is no ethanol smell, filter and dry it, and finally dry it in a 65°C oven for 12 h to constant weight to obtain the pretreated macroporous resin D730; prepare the pretreated macroporous resin D750 by the same method.

(2) Preparation of functionalized resin:

1 g of the macroporous resin D730 pretreated in step (1) was added to 10 mL of 0.2 M sodium phosphate buffer with pH 7.0 containing 0.5% (v/v) glutaraldehyde, and the cross-linking reaction was carried out for 3 h in a water bath shaker at 35° C. and 130 rpm. The functionalized resin was washed with deionized water for 3 to 5 times until the glutaraldehyde smell was no longer detected, thereby obtaining the functionalized macroporous resin D730. The functionalized macroporous resin D750 was prepared in the same manner.

(3) Immobilization:

1 g of the functionalized macroporous resin prepared in step (2) was added to 10 mL of 200 mM, pH 7.0 phosphate buffer containing 1 mg/mL pure enzyme solution of nitrilase AcN-T prepared by the method of Example 2, and adsorbed for 2 h in a water bath shaker at 35°C and 130 rpm. After immobilization, the protein adsorption amount was determined by the method of Example 2. The cross-linked adsorbed macroporous resin was washed with deionized water for 3 to 5 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase by cross-linking-adsorption method, which was recorded as AcN-C@D730 and AcN-C@D750, and then stored in a refrigerator at 4°C for future use.

Example 6: Preparation conditions of immobilized nitrilase

1. Screening of conditions for immobilization of nitrilase by adsorption

(1) Adsorption rate:

After determining to use resin D730 and D750 as immobilized enzyme carriers, 1 g (dry weight) of resin D730 and D750 pretreated by the method of Example 5 were added to 10 mL of pH 7.0, 200 mM sodium phosphate _{( Na2HPO4} - _{NaH2PO4 )} buffer, and then pure enzyme solution of nitrilase AcN-T prepared by the method of Example 2 was added at final concentrations of 0.5 mg/mL, 1.0 mg/mL, and 2.0 mg/mL in terms of protein content, respectively. The mixture was adsorbed on a shaking water bath at 35°C and 130 rpm for 5 h. The adsorption rate was detected by the method of Example 4. The results are shown in Table 2.

The adsorption results are as follows: when the amount of enzyme input is small, the adsorption capacity of both resins has not reached saturation, showing a good adsorption effect, and the protein adsorption efficiency can reach about 95%; when the amount of enzyme input is large, the adsorption on the resin surface or in the pores has reached saturation, and the immobilization efficiency of nitrilase is low.

(2) Specific enzyme activity:

The reaction solution after adsorption for 5 hours in step (1) was filtered, and the filter cake was washed with deionized water for 3 to 5 times, placed on filter paper and dried at room temperature. 1 g of the mixture was used to replace the immobilized nitrilase in the resin-immobilized enzyme activity assay system of Example 3 for enzyme activity assay. The results are shown in Table 2.

It was found that the ideal specific enzyme activity was achieved when the enzyme dosage per gram of D730 and D750 resin was 1.0 mg/mL.

Taking into account the utilization of enzyme protein, a balance is struck between the enzyme immobilization efficiency and the specific enzyme activity of the immobilized enzyme, and the preferred initial enzyme concentration is 10 mg/g resin.

Table 2: Effect of different nitrilase concentrations on the adsorption rate and specific enzyme activity of two types of carriers

(3) Adsorption time

After determining the enzyme loading capacity, the adsorption time curves of the two resins were further explored. In 10 mL of pH 7.0, 200 mM sodium phosphate buffer (Na ₂ HPO ₄ -NaH ₂ PO ₄ ), 1 g (dry weight) of resins D730 and D750 pretreated by the method of Example 5 were added respectively, and then 10 mL of pure enzyme solution of nitrilase AcN-T prepared by the method of Example 2 (final protein concentration 1.0 mg/mL) was added respectively. The mixture was adsorbed for 1, 2, and 3 h in a water bath shaker at 35° C. and 130 rpm. The adsorption rate was detected by the method of Example 4. The results are shown in Table 3.

The results are as follows: When the final concentration of the added enzyme is 1.0 mg/mL, the adsorption efficiency increases rapidly in the early stage of adsorption because the resin adsorption is far from saturation. The adsorption efficiency reaches the highest after 2 to 3 hours, and the nitrilase is basically completely adsorbed by the resin. Therefore, it can be determined that the optimal adsorption time for immobilized nitrilase is 2 to 3 hours.

Table 3: Effect of adsorption time on the adsorption rate of two types of carrier proteins

2. Condition screening of cross-linking-adsorption method

Under the optimal protein loading and adsorption time conditions in step 1, the immobilization method was changed, and the resin surface was first functionalized, and then enzyme molecules were added for cross-linking. Due to the limited pores of the resin, glutaraldehyde with short molecules and simple structure was selected to functionalize the resin surface.

(1) Functionalization reagent concentration

The volume concentration (v/v) of glutaraldehyde added in step 2 of Example 5 was changed to 0.25%, 0.5%, 0.75% and 1%, respectively. The other operations were the same. Functionalization was carried out for 3 hours by adding glutaraldehyde of different concentrations and then enzyme cross-linking was performed. The protein cross-linking amount of the resin after cross-linking and immobilization was determined by the method of Example 2, and the enzyme activity was determined by the method of Example 3.

The results are shown in Table 4. The cross-linking rate of the protein decreased slightly with the increase of the cross-linker concentration. After the resin was treated with four cross-linker concentrations, the enzyme activity was measured under the same amount of immobilized enzyme. When the glutaraldehyde concentration was 0.5%, the immobilized enzyme showed a higher relative enzyme activity compared with the immobilized enzymes treated with other cross-linker concentrations. However, too high a concentration of glutaraldehyde would reduce the enzyme activity of the immobilized enzyme. Therefore, the volume concentration of 0.5% was used as the final concentration of the functionalization reagent.

Table 4: Effects of different cross-linking agent concentrations on carrier type cross-linking rate and enzyme activity recovery rate

(2) Cross-linking time of functionalized reagents

The added volume concentration (v/v) of glutaraldehyde in step 2 of Example 5 was changed to 0.5%, and the cross-linking time of 3h was changed to 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0h respectively. The other operations were the same. The protein adsorption rate was determined by the method of Example 2. The results are shown in Table 5. Compared with the immobilization by adsorption method in step 1 of Example 5, due to the addition of functional groups, the cross-linking rate of the protein was greatly improved compared with the adsorption efficiency. The cross-linking rate can almost reach 100% within 1 to 3h. The preferred cross-linking time is 1 to 3h.

Table 5: Effect of different cross-linking times on the cross-linking rates of two types of carriers

Example 7: Optimal Temperature and Temperature Stability of Immobilized Nitrilase

(1) Optimum temperature

The temperature range of 25-55°C was selected to investigate the effect of different temperatures on the activity of the immobilized enzyme.

1 g (protein loading of 10 mg/g) of immobilized nitrilase AcN@D730, AcN@D750, AcN-C@D730, and AcN-C@D750 prepared by the method of Example 5 were added to 10 mL of pH 7.0, 200 mM sodium phosphate buffer, and incubated at different temperatures (25, 45, and 55° C.) for 10 min, and then the enzyme activity was tested by the method of Example 3. The highest enzyme activity at a certain temperature was qualitatively defined as 100%, and the rest were compared with the highest enzyme activity, which was the relative enzyme activity.

The results are as follows: the optimum temperature of immobilized enzymes AcN@D730 and AcN@D750 prepared by adsorption method can reach 60℃, while the optimum temperature of immobilized enzymes AcN-C@D730 and AcN-C@D750 prepared by adsorption-crosslinking method can reach 70℃.

(2) Thermal stability

First, 1g (protein loading is 10mg/g) of immobilized nitrilase AcN@D730, AcN@D750, AcN-C@D730, AcN-C@D750 prepared by the method of step 2 of Example 5 was added to 10mL of pH 7.0, 200mM sodium phosphate buffer, and placed in a constant temperature water bath at 25°C, 45°C, and 55°C, respectively. Samples were taken at different times (1, 5, 10, 15, 20, 25 days), the original buffer was poured out, new buffer was added, and the enzyme activity was determined by the method of Example 3. The initial enzyme activity was qualitatively 100%, and the rest was compared with the initial enzyme activity to calculate the enzyme activity recovery rate. The enzyme activity recovery rates of AcN-C@D730 and AcN-C@D750 are shown in Table 6.

Results: The immobilized nitrilase prepared by the two immobilization methods (adsorption method, cross-linking-adsorption method) can still maintain more than 90% of its activity after being placed at 25°C, pH 7.0, 200mM sodium phosphate buffer for 25 days. Among them, the cross-linked immobilized enzymes AcN-C@D730 and AcN-C@D750 still have more than 60% of their activity remaining after being placed at 45°C for 20 days, reflecting good thermal stability.

Table 6: Optimal temperature and temperature stability of nitrilase immobilized enzyme

(3) Half-life

The pure enzyme solution of nitrilase AcN-T prepared by the method of Example 2, and the immobilized nitrilase AcN-C@D730 and AcN-C@D750 prepared by the method of Example 5 were placed in a 55°C constant temperature water bath for insulation, and samples were taken at regular intervals to detect the enzyme activity. The activity of the protein when not incubated was used as a control to calculate the relative residual activity (RA) of the protein at each time. With time (h) as the abscissa and the natural logarithm of the relative residual activity (ln(RA)) as the ordinate, a linear fit was performed to obtain the slope k. According to the formula of the first-order inactivation kinetics, the half-life t _1/2 of the enzyme protein can be obtained.

The half-lives of AcN-C@D730 and AcN-C@D750 at 55°C were 480 h and 240 h, respectively, which were extended by 24 and 12 times compared with 20 h of the free enzyme AcN-T.

Example 8: Optimal pH and solution stability of immobilized nitrilase

(1) Optimum pH

Different buffers with a pH range of 6.0 to 8.0 were selected to investigate the effect of different pH on the activity of the immobilized enzyme. The buffer system used was citric acid-sodium citrate buffer (50mM), pH 3.0 to 6.5; phosphate buffer (PBS, 50mM), pH 7.0 to 8.0. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated for comparison.

1 g (protein loading of 10 mg/g) of immobilized nitrilase AcN@D730, AcN@D750, AcN-C@D730, and AcN-C@D750 prepared by the method of Example 5 were placed in 10 mL of different buffer solutions of pH 6.0, pH 7.0, and pH 8.0, respectively, and the enzyme activity was tested by the method of Example 3. The results showed that the four immobilized enzymes achieved an optimal relative enzyme activity of 100% when the pH was 6.0.

Several immobilized enzymes showed higher relative enzyme activities under weak acid (pH=6), while free enzymes showed the highest enzyme activity under pH 7.5. The optimal pH ranges of AcN@D750 and AcN-C@D750 (relative enzyme activity>80%) were narrower, while AcN@D730 and AcN-C@D730 had wider optimal pH ranges, with the optimal pH of AcN-C@D730 being 5-6.

(2) Solution stability

1g (protein loading of 10mg/g) of immobilized nitrilase AcN-C@D730 and AcN-C@D750 prepared by the method of Example 5 were added to 10mL of different buffers of pH 6.0, pH 7.0, and pH 8.0, respectively, and placed at 4°C for 1, 5, 10, 15, 20, and 25 days, and the enzyme activity was tested by the method of Example 3. The initial enzyme activity was qualitatively 100%, and the rest was compared with the initial enzyme activity. The results are shown in Table 7. The stability of several immobilized enzymes in buffer is as follows: AcN-C@D750 shows good stability under pH 6 and pH 7 conditions, and can maintain more than 90% relative enzyme activity after being placed for 25 days. The stability of AcN-C@D730 is slightly weaker, and can only maintain 60% enzyme activity after being placed under pH 6 conditions for 20 days.

Table 7: Optimal pH and solution stability of immobilized nitrilase

Example 9: Protein Desorption of Immobilized Nitrilase

1g (protein loading of 10mg/g) of immobilized nitrilase AcN@D730, AcN@D750, AcN-C@D730, AcN-C@D750 prepared by the method of Example 5 was placed in 10mL 200mM, pH 7.0 sodium phosphate buffer, shaken at 130rpm for 12h in a water bath shaker at 35°C, and the protein content in the solution was determined by the method of Example 2, the protein desorption rate was calculated, and the solution state was observed, and three parallel experiments were repeated. The results are shown in Table 8, and the stability of the cross-linked immobilized enzyme is relatively high, wherein the protein desorption rate of the immobilized nitrilase AcN-C@D730 is the lowest at 1.7%.

Table 8: Protein desorption of immobilized nitrilase

Example 10: Batch reaction of immobilized nitrilase-catalyzed synthesis of 1-cyanocyclohexylacetic acid (1-CA) (1) Batch reaction at low substrate concentration

1 g (protein loading of 10 mg/g) of immobilized nitrilase (AcN@D730, AcN@D750, AcN-C@D730, AcN-C@D750) prepared by the method of Example 5 was added to 10 mL, 200 mM, pH 7.0 sodium phosphate buffer, 0.2 M substrate 1-cyanocyclohexylacetonitrile (1-CN) was added, and the mixture was placed in a water bath shaker at 35°C and 150 rpm for reaction.

Reaction batch: A reaction batch was defined as one with a conversion rate exceeding 90%.

After each reaction, the reaction solution was collected, and the immobilized enzyme was washed 3 to 5 times with deionized water before the next batch of catalytic reaction was carried out. During the reaction, the substrate concentration was detected by the method of Example 3, and the substrate conversion rate was calculated. After a batch of reactions was completed, the product concentration was detected and the product yield was calculated. The results are shown in Table 9. The cross-linked immobilized enzymes AcN-C@D750 and AcN-C@D730 were catalyzed for 2h per batch, and the substrate conversion rate could still reach 100% after 30 batches. The adsorbed immobilized enzymes AcN@D750 and AcN@D730 had a catalytic conversion rate of 20 batches (2h per batch), and the cross-linked immobilized enzymes had a stronger reusability.

Table 9: Batch reactions catalyzed by nitrilase immobilized enzymes

(2) High substrate concentration batch reaction

In order to verify the catalytic effect of immobilized enzyme on high concentration substrate, the substrate concentration in step 1 was changed to 1 M, the amount of immobilized enzyme was 100 g/L (the protein loading was 10 mg/g), and other conditions remained unchanged. The results are shown in Table 10. 100 g/L of AcN@D750 and AcN@D730 can convert about 98% of the substrate within 7 hours, and AcN-C@D750 and AcN-C@D730 can convert about 96% of the substrate within 5 hours.

The immobilized nitrilase in step 1 was replaced with 10 g/L wet bacteria AcN-T prepared by the method of Example 1, with an enzyme content of about 1600 mg/L. Other operations were the same as those of the free cell control.

Compared with free cells, currently in practice, 10g/L of free cells are used as catalysts, and the enzyme content is about 1600mg/L, while the equivalent enzyme dosage of immobilized enzyme is only 1000mg/L, and it can be reused for dozens of batches. Immobilization greatly improves the catalytic efficiency and reusability of the enzyme. The optimal cross-linked immobilized nitrilase obtained can still achieve a substrate conversion rate of 100% after catalysis of 30 batches.

Table 10: Batch reactions of high-concentration substrates catalyzed by immobilized nitrilase

Example 11: Storage stability of immobilized nitrilase

The storage stability of resin-immobilized nitrilase is excellent, exceeding that of most immobilized enzyme preparations reported so far.

The AcN-C@D730 and AcN-C@D750 recovered after 30 batches of reactions in Example 10 were transferred to a 4°C refrigerator and stored for 30 days before being taken out. They could still completely convert 200 mM substrate within two hours.

0.1 g (protein loading of 10 mg/g) of AcN-C@D730 and AcN-C@D750 prepared by the method of Example 5 was placed in 1 mL of 200 mM pH 7.0 sodium phosphate buffer, with a final addition amount of 100 g/L of immobilized enzyme. It was stored at the optimal temperature (70°C) and optimal pH 6 for 30 days. The results are shown in Table 11. The residual enzyme activity can reach more than 90%.

The immobilized nitrilase AcN@D730, AcN@D750, AcN-C@D730, and AcN-C@D750 prepared by the method of Example 5 were dried in the shade, placed in an environment of 4°C (stored in an EP tube after drying at room temperature) for 6 months, and then the enzyme activity was determined. The results are shown in Table 11. The residual enzyme activity of AcN@D730 reached 66.67%, while the residual enzyme activity of AcN-C@D730 reached 80.23%. Under the same conditions, the residual enzyme activity of AcN@D750 only reached 28.57%, and AcN-C@D750 had only about 19.71% residual enzyme activity. Although the catalytic performance and storage stability of these two resins after cross-linking treatment are relatively good, the immobilized enzyme based on D730 resin performs better in storage performance.

Table 11: Storage stability of immobilized nitrilase

Claims (10)

1. A method for immobilizing nitrilase to improve enzymatic performance, characterized in that the immobilization method is to prepare immobilized nitrilase by using an adsorption method or a cross-linking-adsorption method, using a macroporous resin as a carrier and a pure enzyme solution obtained by fermentation and cultivation of an engineered bacterium containing a nitrilase encoding gene as an active component; the nucleotide sequence of the nitrilase encoding gene is shown in SEQ ID NO.1. 2. The method according to claim 1, characterized in that the adsorption method is carried out according to the following steps: immersing the macroporous resin in a pure enzyme solution, adsorbing and immobilizing for 1 to 3 hours in a water bath under the conditions of 25 to 55° C. and 100 to 200 rpm, filtering, washing the filter cake with deionized water for 3 to 5 times, placing it on filter paper and drying it at room temperature to obtain immobilized nitrilase. 3. The method according to claim 2, wherein the pure enzyme solution is used in an amount of 5-15 mg/g resin based on protein content. 4. The method according to claim 1, characterized in that the cross-linking-adsorption method is carried out according to the following steps: the macroporous resin is added to a sodium phosphate buffer containing glutaraldehyde at a volume concentration of 0.25-1.00%, cross-linked for 0.5-3h at 25-55°C and 100-200rpm on a shaking table, and washed with deionized water for 3-5 times until glutaraldehyde is no longer detected; then pure enzyme solution is added, and adsorption and immobilization are carried out for 1-3h in a water bath under the conditions of 25-55°C and 100-200rpm, filtered, and the filter cake is washed with deionized water for 3-5 times, and placed on filter paper to dry at room temperature to obtain immobilized nitrilase. 5. The method according to claim 4, characterized in that the volume of the phosphate buffer containing glutaraldehyde is 5-15 mL/g based on the mass of the macroporous resin; and the amount of the pure enzyme solution is 5-15 mg/g resin based on the protein content. 6. The method according to claim 1, characterized in that the macroporous resin model is one of the following: AB-8, 201*7, D001, D113, D115, D201, D730, D750, HPD750. 7. The method according to claim 1 is characterized in that the macroporous resin is first activated and pretreated before immobilization: the macroporous resin is added to an ethanol aqueous solution with a volume concentration of 75-95%, immersed at 25°C for 12 hours, then washed with deionized water for 3-5 times until there is no ethanol smell, filtered, and the filter cake is dried in an oven at 65°C to constant weight to obtain the pretreated macroporous resin. 8. The method according to claim 1, characterized in that the pure enzyme solution is prepared as follows: the recombinant Escherichia coli containing the nitrilase encoding gene is inoculated into a test tube containing LB liquid culture medium with a final concentration of 50 μg/mL kanamycin resistance, and cultured at 37°C and 180 rpm for 8 to 12 hours, and then inoculated into a fresh LB liquid culture medium shake flask containing a final concentration of 50 μg/mL kanamycin resistance at an inoculum concentration of 1% by volume, and cultured in a shaker at 37°C and 150 rpm for 2 to 3 hours until the bacterial OD600 reaches 0.6 to 0.8, and then IPTG with a final concentration of 0.1 mM is added, and after induction culture at 28°C for 8 to 12 hours, the supernatant is discarded and the wet bacterial cells are collected; 0.9 g of the wet bacterial cells are taken and suspended in 30 mL pH 7.0, 200mM sodium phosphate buffer, in an ice bath, ultrasonically disrupted for 30min under the conditions of a disrupting power of 240W, disrupting for 1s and stopping for 2s, centrifuged at 4°C and 12000rpm for 10min, and the supernatant was taken as the crude enzyme solution; the crude enzyme solution was purified by Ni column with a sample flow rate of 1.5mL/min, and the impurity protein was eluted with pH 6-8, 200mM sodium phosphate buffer containing 50mM imidazole until the ultraviolet detection signal decreased to the baseline, the Ni column was balanced with pH 6-8, 200mM sodium phosphate buffer containing 50mM imidazole, eluted for 5 column volumes, and then eluted with pH 6-8, 200mM sodium phosphate buffer containing 500mM imidazole, and the elution peak was collected according to the signal change of the ultraviolet detector, and the collected solution was dialyzed and purified using a dialysis bag with a molecular weight cutoff of 14kDa, and the intercepted liquid was taken to obtain a pure nitrilase enzyme solution. 9. Use of the immobilized nitrilase prepared by the method of claim 1 in the preparation of 1-cyanocyclohexylacetic acid. 10. The use according to claim 9, characterized in that the method of the use is: using immobilized nitrilase as a catalyst, 1-cyanocyclohexylacetonitrile as a substrate, and pH 7.0, 200mM sodium phosphate buffer as a reaction medium to form an aqueous phase reaction system, reacting at 25-55°C and 110-150rpm for 1-3h, after the reaction is complete, filtering the mixed solution to recover the immobilized enzyme, separating and purifying the filtrate to obtain the product 1-cyanocyclohexylacetic acid; in the aqueous phase reaction system, the substrate is added at a concentration of 0.2-1M, and the amount of immobilized nitrilase is 20-200g/L; the protein loading of the immobilized nitrilase is 0.50-10mg/g.