Preparation method and application of surface charge modified enzyme mutant and adaptive immobilized enzyme

Abstract

The invention discloses a preparation method and application of a surface charge modified enzyme mutant and an adaptive immobilized enzyme. The enzyme is subjected to surface charge modification mutation and then is combined with MOFs to form the immobilized enzyme, so that the immobilized enzyme has better enzyme activity recovery rate, stability and reusability, and efficient synthesis of medicine or chemical intermediates can be realized; the intermediate can be used as a gabapentin intermediate, such as 1-cyanocyclohexyl acetic acid (nitrilase), a clopidogrel key intermediate (S)-chlorophenylglycine (esterase, acylase), a chemical intermediate (S)-1-methoxy-2-propylamine (transaminase) and the like; or efficient degradation of toxic substances, such as ricinine in castor bean meal, is realized.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the preparation method and application of immobilized enzymes based on surface charge-modified enzyme mutants and MOFs. It includes various surface charge-modified enzyme mutants, immobilized enzymes formed by combining enzyme mutants with metal-organic frameworks (MOFs), their preparation methods, and the applications of these immobilized enzymes in biocatalysis. (II) Background Technology

[0002] Metal-organic frameworks (MOFs), as a class of porous crystalline materials assembled from metal ions / clusters and organic ligands through coordination bonds, have become ideal carriers for immobilized enzymes due to their high specific surface area, tunable pore structure, abundant active sites, and good chemical stability. However, the compatibility between enzyme molecules and immobilized carriers needs to be improved.

[0003] To improve the compatibility between enzyme molecules and carrier materials, enzyme molecules can be further modified or their surfaces modified. Common enzyme molecule modification strategies include active site modification, substrate pocket remodeling, and distal site modification. However, these strategies focus on altering the catalytic activity, stability, and tolerance of the enzyme molecule itself, and lack relevance to immobilization technology.

[0004] By modifying the local surface charge of the enzyme to the opposite charge to that of the MOF carrier, the electrostatic interaction between the enzyme and the carrier is enhanced in a targeted manner, which significantly improves the effective adsorption of the enzyme inside the MOF structure, avoids local aggregation, and increases the enzyme loading per unit mass of carrier. However, the bottleneck lies in the location of the enzyme surface charge modification. (III) Summary of the Invention

[0005] The purpose of this invention is to provide surface-charge modified enzyme mutants, a method for preparing adaptable immobilized enzymes, and their applications. By modifying the enzyme with surface charge and then combining it with MOFs to form immobilized enzymes, these enzymes exhibit better enzyme activity recovery, stability, and reusability, enabling the efficient synthesis of pharmaceutical or chemical intermediates, such as the gabapentin intermediate 1-cyanocyclohexylacetic acid (nitrile hydrolase) and the key intermediate of clopidogrel (…). S )-o-chlorophenylglycine (esterase, acylase), chemical intermediate ( S It can be used to degrade toxic substances such as 1-methoxy-2-propanamine (transaminase) or ricinole in castor meal.

[0006] The technical solution adopted in this invention is:

[0007] In a first aspect, the present invention provides an enzyme mutant based on surface charge modification. The enzyme mutant is obtained by mutating two adjacent amino acid residues on the enzyme surface, far from the enzyme's active site, thereby altering the local surface charge of the enzyme and enhancing its compatibility with the immobilization carrier. The selection criteria for the two adjacent amino acid residues are: they originally did not carry the charge to be modified (e.g., changing from originally negative to negative); they are on the protein surface and far from the enzyme's active site; and the two amino acid residues are adjacent (simultaneous charge modification is sufficient to alter the local charge distribution).

[0008] Furthermore, the enzyme mutants include nitrile hydrolase mutant AcN-T2 (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2), esterase BS-M2 (nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4), acylase SM-M2 (nucleotide sequence as shown in SEQ ID NO.5, amino acid sequence as shown in SEQ ID NO.6), transaminase BM-M2 (nucleotide sequence as shown in SEQ ID NO.7, amino acid sequence as shown in SEQ ID NO.8), and nitrile hydratase NHasePs-M2 (nucleotide sequence as shown in SEQ ID NO.9, amino acid sequence as shown in SEQ ID NO.10).

[0009] Secondly, the present invention provides an MOF-immobilized enzyme of the enzyme mutant, wherein the immobilized enzyme is prepared using MOFs as a carrier and crude enzyme solution extracted from engineered bacteria expressing the enzyme mutant after induction culture as the active component under magnetic stirring at 4°C.

[0010] Furthermore, the MOFs include negatively charged MIL-101 (Cr) carrier materials or positively charged ZIF-8.

[0011] Furthermore, the method for preparing the crude enzyme solution is as follows: the engineered bacteria containing the enzyme mutant encoding gene are induced and cultured to obtain wet bacterial cells, which are then suspended in a pH 6-8 sodium phosphate buffer (preferably 200 mM, pH 7.0), ultrasonically disrupted, centrifuged, and the supernatant is collected to obtain the crude enzyme solution; the ultrasonic disruption frequency is 1 second for 2 seconds, the power is 240 W, and the time is 30 min.

[0012] Furthermore, the protein concentration of the crude enzyme solution is 5-10 mg / mL, preferably 5 mg / mL.

[0013] Furthermore, the preparation method of MOF-immobilized enzyme is as follows: MOF powder is added to a buffer solution with pH 2-8 (preferably pH 7-8), crude enzyme solution of mutant enzyme is added, and the reaction is carried out at 4°C with magnetic stirring for 6-8 h to ensure sufficient contact between the enzyme and the carrier; after the reaction, the immobilized enzyme and unimmobilized enzyme protein are separated by centrifugation at 6000 rpm for 5 min; finally, the precipitate is washed with ultrapure water and dried at 25°C to obtain the immobilized enzyme; the protein content of the mutant enzyme to the mass ratio of MOFs is 1:25-30 (preferably 1:22-25); the volume of the buffer solution is 1-5 mL / mg (preferably 3 mL / mg) based on the carrier mass.

[0014] Thirdly, the present invention provides an application of the MOFs-immobilized enzyme in the preparation of compound intermediates.

[0015] Furthermore, the compound intermediates include gabapentin intermediate 1-cyanocyclohexylacetic acid (nitrile hydrolase) and clopidogrel key intermediate ( S )-o-chlorophenylglycine (esterase, acylase), chemical intermediate ( S 1-Methoxy-2-propylamine (transaminase).

[0016] Furthermore, the application method is as follows: using MOF-immobilized enzymes as catalysts, adding substrates, and using purified water or pH 7-8 sodium phosphate buffer as the reaction medium to form a reaction system, reacting at 30-35℃ and 600-800 rpm to obtain a reaction solution containing the product. The reaction solution is then separated and purified, and the catalyst is recovered for reuse to obtain the intermediate; the substrate includes 1-cyanocyclohexylacetonitrile, R,S -O-chlorophenylglycine methyl ester, N -phenylacetyl-( R,S The catalyst is added at a final concentration of 1-5 g / L; the substrate is added at a final concentration of 200-1000 mM; when the substrate is 1-methoxy-2-propanone, isopropylamine is added as a co-substrate, and pyridoxal phosphate (PLP) is added as a coenzyme, the molar ratio of 1-methoxy-2-propanone to isopropylamine is 1:1.5, and the final concentration of PLP is 1 mM.

[0017] Furthermore, when the MOF-immobilized enzyme is a nitrile hydrolase, it is used to prepare the gabapentin intermediate 1-cyanocyclohexylacetic acid. The preparation steps are as follows: using the MOF-immobilized enzyme (AcN-T2@ZIF-8) as a catalyst, 1-cyanocyclohexylacetonitrile as a substrate, and purified water or 200 mM, pH 7.0 sodium phosphate buffer as the reaction medium, the reaction is carried out at 35°C and 600 rpm to obtain a reaction solution containing the product 1-cyanocyclohexylacetic acid. The reaction solution is separated and purified, and the catalyst is recovered and reused to obtain 1-cyanocyclohexylacetic acid. The final concentration of the catalyst added is 1-5 g / L, preferably 2.5 g / L; the final concentration of the substrate added is 200-1000 mM, preferably 500 mM.

[0018] Furthermore, when MOFs immobilize an esterase, it is used to prepare clopidogrel intermediates. S In 1-o-chlorophenylglycine, the preparation steps are as follows: an enzyme (BS-M2@MIL-101) catalyst is immobilized in MOFs, and... R,S Using methyl 2-o-chlorophenylglycine as a substrate, a reaction system was constructed with 200 mM, pH 8.0 sodium phosphate buffer as the reaction medium. The reaction was carried out at 35°C and 600 rpm. The catalyst was recovered and reused to obtain the product. S )-o-chlorophenylglycine. The catalyst is added to a final concentration of 1-5 g / L, preferably 3 g / L; the substrate is added to a final concentration of 100-500 mM, preferably 200 mM.

[0019] Furthermore, when MOFs immobilize enzymes as acylases, they are used to prepare clopidogrel intermediates. S )-o-chlorophenylglycine, the preparation steps are as follows: immobilize enzyme (SM-M2@MIL-101) catalyst with MOFs, and then... N -phenylacetyl-( R,S Using 1,4-chlorophenylglycine as a substrate, a reaction system was constructed with purified water or 200 mM, pH 7.0 sodium phosphate buffer as the reaction medium. The reaction was carried out at 35°C and 600 rpm to obtain a product containing ( S )-o-chlorophenylglycine and residual substrate N -phenylacetyl-( R The reaction solution of )-o-chlorophenylglycine; the reaction solution is separated and purified, the catalyst is recovered and reused, and ( S )-o-chlorophenylglycine. The final concentration of the catalyst added is 1-5 g / L, preferably 3.5 g / L; the final concentration of the substrate is 100-500 mM, preferably 200 mM.

[0020] Furthermore, when MOFs immobilize transaminases, they are used to prepare ( SThe preparation steps for 1-(+)-1-methoxy-2-propylamine are as follows: Using an enzyme immobilized in MOFs (BM-M2@MIL-101) as a catalyst, 1-methoxy-2-propanone and isopropylamine as substrates, pyridoxal phosphate (PLP) as a coenzyme, and 200 mM, pH 7.8 sodium phosphate buffer as the reaction medium, the reaction is carried out at 30℃ and 800 rpm to obtain the product (+)-1-methoxy-2-propylamine. S The reaction solution of )-(+)-1-methoxy-2-propane, etc.; the reaction solution is separated and purified, the catalyst is recovered and reused, and ( S The catalyst is 1-(+)-1-methoxy-2-propane; the final concentration of the catalyst is 1-5 g / L, preferably 3.2 g / L; the final concentration of the substrate 1-methoxy-2-propanone is 200-1000 mM, preferably 600 mM; the molar ratio of 1-methoxy-2-propanone to isopropylamine is 1:1.5; and the final concentration of pyridoxal phosphate is 1 mM.

[0021] Fourthly, this invention also provides an application of MOF-immobilized enzyme in the degradation of ricinole in castor bean meal. The application is carried out according to the following steps: using MOF-immobilized enzyme (NHasePs-M2@ZIF-8) as a catalyst and castor bean meal as a substrate, a reaction system is formed by adding 200 mM, pH 7.5 sodium phosphate buffer. The system is then cultured at 37°C and 300 rpm to achieve the degradation of ricinole. The mass ratio of catalyst to substrate is 1:80-100 (preferably 1:100), and the amount of buffer added to the castor bean meal is 100-1000 mL / kg, preferably 200 mL / kg.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] (1) This invention selects to directionally modify the charge on the enzyme surface, and regulates the local charge distribution on the enzyme molecule surface through site-directed mutagenesis to optimize enzyme stability. The modified mutant is immobilized with MOFs to enhance the compatibility between the enzyme molecule and the immobilization carrier, and enhance the stability of the enzyme during the construction of MOFs and within the structure; it can also improve the binding efficiency between the enzyme and the carrier through charge complementarity, thereby improving catalytic activity and enzyme activity recovery rate.

[0024] (2) The present invention uses crude enzyme solution to prepare MOF immobilized enzymes. The preparation process is simple, reduces costs, and is easier to scale up for production without relying on equipment. The average immobilization rate reaches more than 80%, and the enzyme activity recovery rate reaches more than 70%, with both the immobilization rate and enzyme activity recovery rate being high.

[0025] (3) The AcN-T2@ZIF-8 prepared according to the method of the present invention can completely convert 200 mM substrate within 3 h, completely convert 500 mM substrate within 6 h, and basically completely convert 1000 mM substrate within 11 h.

[0026] The BS-M2@MIL-101 prepared according to the method of the present invention can completely resolve 100 mM substrate within 4 h, completely resolve 200 mM substrate within 8 h, and substantially completely resolve 500 mM substrate within 12 h.

[0027] The SM-M2@ZIF-8 prepared according to the method of the present invention can completely resolve 100 mM substrate within 12 h, completely resolve 200 mM substrate within 24 h, and substantially completely resolve 500 mM substrate within 40 h.

[0028] The BM-M2@MIL-101 prepared according to the method of the present invention can completely convert 200 mM substrate within 6 h, completely convert 600 mM substrate within 24 h, and substantially completely convert 1000 mM substrate within 36 h.

[0029] The NHasePs-M2@ZIF-8 prepared according to the method of the present invention can basically detoxify 1 kg of substrate in 12 h.

[0030] (4) The AcN-T2@ZIF-8 prepared in this invention can completely convert 200 mM substrate in 25 batches of batch reaction for the preparation of 1-cyanocyclohexylacetic acid, demonstrating excellent reusability and stability. (iv) Description of the attached drawings

[0031] Figure 1 This is a flowchart illustrating the preparation process of enzymes immobilized in MOFs.

[0032] Figure 2 This is a reaction process diagram for the synthesis of 1-CA catalyzed by AcN-T2@ZIF-8.

[0033] Figure 3 Synthesis of BS-M2@MIL-01 catalyzed by ( S )-O-chlorophenylglycine reaction process diagram.

[0034] Figure 4 Synthesis of SM-M2@ZIF-8 catalyzed by ( S )-o-chlorophenylglycine reaction process diagram.

[0035] Figure 5 Synthesis of BM-M2@MIL-101 catalyzed by ( S )-(+)-1-methoxy-2-propane reaction process diagram.

[0036] Figure 6 This is a reaction process diagram of the catalytic degradation of ricinine in castor bean meal by NHasePs-M2@ZIF-8. (V) Detailed Implementation Methods

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0038] Example 1: Construction of genetically engineered bacteria expressing mutants

[0039] 1) Construction of Escherichia coli genetically engineered bacteria expressing the nitrile hydrolase mutant AcN-T2:

[0040] Using the DNA of nitrile hydrolase AcN-T as shown in SEQ ID NO.1 of patent CN111172140A as a template, PCR amplification was performed using primers 1 and 2 (see Table 1 for details) with high-fidelity Pfu DNA polymerase to obtain the gene sequence of nitrile hydrolase AcN-T. The pET-28a(+)-AcN-T plasmid was constructed and transformed into the host bacteria. E. coli BL21(DE3), an engineered bacterium expressing nitrile hydrolase. E. coli BL21(DE3) / pET-28a(+)-AcN-T.

[0041] The surface charge of the nitrile hydrolase AcN-T was directionally modified using site-directed mutagenesis. The mutation sites were selected at arginine 138 and threonine 139, far from the active site. Specific primers were designed, and PCR amplification yielded a recombinant plasmid containing the R138E / T139E mutant (AcN-T2, nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2), which was then transformed into... E. coli Positive clones were screened after BL21(DE3) competent cells were used. Following the method in Example 2, the enzyme was induced to express by IPTG, purified by nickel column affinity chromatography, and dialyzed to obtain crude enzyme solution and high-purity mutant AcN-T2 enzyme solution.

[0042] Table 1: Primer design for nitrile hydrolases and mutant genetically engineered bacteria

[0043]

[0044] 2) Construction of Escherichia coli genetically engineered bacteria expressing the esterase mutant BS-M2:

[0045] Based on data from Bacillus subtilis in GenBank ( Bacillus subtilisThe wild-type esterase (GenBank: ATCC 1022, U46134.2, estB, AAC44253.1) was amplified using primers 1 and 2 in Table 2. The pET-28a(+)-BS plasmid was constructed and transformed into the host bacteria. E. coli BL21(DE3), an engineered bacterium expressing wild-type esterase BS. E. coli BL21(DE3) / pET-28a(+)-BS, this wild-type esterase is denoted as esterase BS.

[0046] The surface charge of the esterase was modified using site-directed mutagenesis. The alanine residue at position 96 and the serine residue at position 97, located far from the active site and adjacent to it, were selected as double mutation sites. Specific primers were designed (Table 2), and PCR amplification was performed to obtain a recombinant plasmid containing the A96K / S97R mutant (BS-M2, nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4). This plasmid was then transformed into... E. coli Positive clones were screened after BL21(DE3) competent cells were used. Following the method in Example 2, the enzyme was induced to express by IPTG, purified by nickel affinity chromatography, and dialyzed to obtain crude enzyme solution and high-purity double mutant esterase solution.

[0047] Table 2: Primer design for esterases and mutant genetically engineered bacteria

[0048]

[0049] 3) Construction of Escherichia coli genetically engineered bacteria expressing acylase SM-M2:

[0050] Using the SM genomic DNA shown in SEQ ID NO.5 of patent CN118325877A as a template, the acylase SM was amplified using primers 1 and 2 in Table 3 to construct the pET-28b(+)-SM plasmid, which was then transformed into the host bacteria. E. coli BL21(DE3), constructing engineered bacteria E. coli BL21(DE3) / pET-28b(+)-SM, denoted as acylase SM.

[0051] The surface charge of the acylase was modified using site-directed mutagenesis. Valine (position 132) and leucine (position 133), located far from the active site and adjacent to it, were selected as double mutation sites. Specific primers were designed (Table 3), and PCR amplification yielded a recombinant plasmid containing the V132D / L133E mutant (SM-M2, nucleotide sequence as shown in SEQ ID NO. 5, amino acid sequence as shown in SEQ ID NO. 6). This plasmid was then transformed into... E. coliPositive clones were screened after BL21(DE3) competent cells were used. Following the method in Example 2, the enzyme was induced to express by IPTG, purified by nickel column affinity chromatography, and dialyzed to obtain crude enzyme solution and high-purity double mutant acylate solution.

[0052] Table 3: Primer design for acylases and mutant genetically engineered bacteria

[0053]

[0054] 4) Construction of Escherichia coli genetically engineered bacteria expressing transaminase BM-M2:

[0055] The sequence accession number 5G09 (gene sequence WP_015810478.1) was found in the gene bank, originating from... Bacillus megaterium of( S )-Selective ω-transaminase. Bacillus megaterium Using the genome as a template, primers 1 and 2 (Table 4) were used to amplify the transaminase BM and transform it into a vector to obtain the pET-28a(+)-BM plasmid, which was then transformed into the host bacteria. E. coli BL21(DE3), constructing engineered bacteria E. coli BL21(DE3) / pET-28a(+)-BM, denoted as transaminase BM, was used to construct a transaminase mutant.

[0056] The surface charge of transaminase BM was modified using site-directed mutagenesis. Valine (position 108) and threonine (position 109), located far from the active site and adjacent to it, were selected as double mutation sites. Specific primers were designed (Table 4), and PCR amplification yielded a recombinant plasmid containing the V108K / T109R mutant (BM-M2, nucleotide sequence as shown in SEQ ID NO.7, amino acid sequence as shown in SEQ ID NO.8). This plasmid was then transformed into... E. coli Positive clones were screened after BL21(DE3) competent cells were used. Following the method in Example 2, the enzyme was induced to express by IPTG, purified by nickel column affinity chromatography, and dialyzed to obtain crude enzyme solution and high-purity double mutant transaminase solution.

[0057] Table 4: Primer Design for Transaminases and Mutant Genetically Engineered Bacteria

[0058]

[0059] 5) Construction of Escherichia coli expressing nitrile hydratase NHasePs:

[0060] Based on nitrile hydratase (GenBank: QFR55171.1), the target gene was amplified using primers 1 and 2, and the pET-28b(+)-NHasePs plasmid was constructed and transformed into the host bacteria. E. coli BL21(DE3), constructing engineered bacteria E. coli BL21(DE3) / pET-28b(+)-NHasePs, denoted as nitrile hydratase NHasePs.

[0061] The surface charge properties of nitrile hydratase NHasePs were modified using site-directed mutagenesis. The 98th and 99th lysine residues, located far from the active site and adjacent to it, were selected as double mutation sites. Specific primers (Table 5) were designed, and PCR amplification yielded a recombinant plasmid containing the K98E / H99E mutant (NHasePs-M2, nucleotide sequence as shown in SEQ ID NO. 9, amino acid sequence as shown in SEQ ID NO. 10). This plasmid was then transformed into... E. coli Positive clones were screened after BL21(DE3) competent cells were used. Following the method in Example 2, the enzyme was induced to express by IPTG, purified by nickel column affinity chromatography, and dialyzed to obtain crude enzyme solution and high-purity double mutant nitrile hydratase solution.

[0062] Table 5: Primer design for nitrile hydratase and mutant genetically engineered bacteria

[0063]

[0064] Example 2: Preparation of crude enzyme solution and pure enzyme

[0065] (1) Weigh 0.9 g of each of the wet bacterial cells AcN-T2, BS-M2, SM-M2, BM-M2, and NHasePs-M2 obtained in Example 1, add them to 30 mL of sodium phosphate buffer (200 mM, pH 7.0) and mix thoroughly. Keep the bacterial suspension in an ice bath to prevent enzyme activity loss. Place it in an ultrasonic cell disruptor, adjust the disruption frequency to 1 s disruption followed by 2 s pause, the power to 240 W, and the disruption time to 30 min. Stop disruption when the solution becomes relatively transparent. Place the disrupted solution in a 50 mL centrifuge tube and centrifuge at 12000 rpm and 4℃ for 10 min. Discard the precipitate and collect the supernatant to obtain the corresponding crude enzyme solution.

[0066] (2) Preparation of pure enzymes

[0067] The crude enzyme solution was purified using a Ni column at a flow rate of 1.5 mL / min. Impurities were eluted with a pH 6-8, 200 mM sodium phosphate buffer containing 50 mM imidazole until the UV detection signal returned to baseline. The Ni column was then equilibrated with a pH 6-8, 200 mM sodium phosphate buffer containing 50 mM imidazole, and eluted for 5 column volumes. Elution was then performed again with a pH 6-8, 200 mM sodium phosphate buffer containing 500 mM imidazole. The elution peak was collected based on the change in UV detector signal. The collected solution was purified by dialysis (dialysis bag molecular weight cutoff 14 kDa, dialysis buffer 50 mM, pH 7 sodium phosphate buffer). The cutoff solution was collected to obtain the pure enzyme solution.

[0068] (3) Determination of protein content: Coomassie brilliant blue method

[0069] Preparation of Coomassie Brilliant Blue Detection Solution: Weigh 10 mg of Coomassie Brilliant Blue G-250, dissolve it in 5 mL of 95% ethanol, add 10 mL of 85% phosphoric acid, and dilute to 100 mL with pure water. Store in a brown bottle for later use.

[0070] Preparation of standard curves for enzyme protein concentrations: Weigh bovine serum albumin into 10 mL EP tubes and prepare gradient solutions with final concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / mL. Take 5 μL of each gradient standard solution and each diluted sample to be tested into the deep wells of an ELISA plate, add 200 μL of Coomassie Brilliant Blue detection solution, mix thoroughly, and measure the absorbance at 595 nm using an ELISA reader within 10 min. Plot the standard curve for the standard solutions and determine the concentration of the sample to be tested.

[0071] Example 3: Determination of enzyme activity

[0072] (1) Definition of enzyme activity

[0073] The activity of nitrile hydrolase AcN-T is defined as the amount of enzyme required to catalyze the production of 1 μmol of 1-cyanocyclohexylacetonitrile per minute at 35℃ and pH 7.0, defined as 1 U.

[0074] The activity of esterase BS is defined as the rate of catalysis per minute at 35°C and pH 8.0. R,S -o-chlorophenylglycine methyl ester generates 1 μmol of ( S The amount of enzyme required to obtain 1 U of 1-o-chlorophenylglycine is defined as 1 U.

[0075] The activity of acylase SM is defined as the rate of catalysis per minute of N-phenylacetyl-( R,S )-o-chlorophenylglycine generates 1 μmol of ( S The amount of enzyme required to obtain 1 U of 1-o-chlorophenylglycine is defined as 1 U.

[0076] The enzyme activity of transaminase BM is defined as the rate of synthesis of 1 μmol ( ) per minute under conditions of 30°C and pH 7.8. S The amount of enzyme required for 1 U of 1-(+)-1-methoxy-2-propane is defined as 1 U.

[0077] The enzyme activity of nitrile hydratase (NHasePs) is defined as the amount of enzyme required to catalyze the degradation of 1 μmol of ricinine per minute at 37°C and pH 7.5, which is defined as 1 U.

[0078] (2) Detection methods for the product

[0079] The liquid chromatography method for detecting the product 1-cyanocyclohexylacetic acid was as follows: a Shimadzu SPD-16 liquid chromatograph was used with an XBridge BEH C18 column (130 Å, 5 μm, 4.6 mm × 250 mm, Waters); the mobile phase was 76% aqueous buffer (0.58 g / L NH4H2PO4 and 2.1 g / L NaClO4, pH 1.84) and 24% acetonitrile; the UV detector wavelength was 215 nm; the column temperature was 40℃; the flow rate was 1.0 mL / min; the injection volume was 10 μL; and the analysis time was 20 min.

[0080] product( S Detection of 1-o-chlorophenylglycine: A Thermo Fisher Scientific Ultimate 3000 HPLC system was used. The column was a J&K Scientific C18-H column (4.6 × 250 mm, 5 μm). The mobile phase was acetonitrile:water (0.1% perchloric acid) = 1:1 (V:V); the flow rate was 0.7 mL / min; the detection wavelength was 220 nm; and the column temperature was 30℃. Retention time: ( S )-o-chlorophenylglycine 4.1 min; phenylacetic acid 7.2 min; N-phenylacetyl-( R,S )-o-chlorophenylglycine 9.8 min.

[0081] product( SDetection of 1-fluoro-2-propanamine: A Thermo Fisher Scientific Ultimate 3000 HPLC system was used. The column was a ChromCorePEP (5 μm, 4.6 mm × 250 mm); the mobile phase was water + 0.1% trifluoroacetic acid; the detection wavelength was 340 nm; the flow rate was 0.8 mL / min; and the column temperature was 25℃. Derivatization reagent: 0.004 g of 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (Marfey reagent) was weighed and dissolved completely with 1 mL of acetonitrile. Derivatization reaction and determination: Add 40 μL of derivatization reagent, 72 μL of 1 M sodium bicarbonate aqueous solution, and 200 μL of DMSO to 40 μL of the reaction solution sample. Place in a metal bath at 40℃ and shake at 800 rpm for 2 h. Afterward, terminate the reaction with 80 μL of 1 M hydrochloric acid. Centrifuge, pass the supernatant through a 0.22 μm membrane, and inject 10 μL for analysis. The analysis time is 15 min. Retention time: ( S )-1-methoxy-2-propane 9 min.

[0082] Detection of ricin: Ricin was detected using ultraviolet spectrophotometry. The specific steps are as follows: Accurately weigh 10.0 g of castor seed cake powder, add 100 mL of chloroform, reflux for 12 hours, filter and collect the filtrate; concentrate the filtrate to dryness using a rotary evaporator, wash the residue three times with 100 mL of anhydrous diethyl ether, and then dry it thoroughly in a vacuum desiccator; dissolve the dried residue in methanol and dilute to a volumetric flask of 100 mL, mix well and filter, take 1.0 mL of the filtrate and dilute to a volumetric flask of 10 mL; finally, use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 313 nm, and calculate the ricin content according to the pre-established standard curve.

[0083] (3) Methods for detecting enzyme activity

[0084] Free enzyme activity (U f Detection: Take 100 μL of the crude enzyme solution obtained in Example 2 and mix it with 900 μL of sodium phosphate buffer (200 mM, pH 7.0) to form a 1 mL system. Incubate in a metal bath at 35℃ and 800 rpm for 10 min. After incubation, add the substrate and continue the reaction for 10 min. After the reaction is completed, add a small amount of 6 M hydrochloric acid solution to terminate the reaction. Centrifuge at 12000 rpm for 1 min and take the supernatant. Dilute it 10 times and perform liquid chromatography analysis.

[0085] Immobilized enzyme activity (U IDetection: After the immobilized enzyme is air-dried at room temperature, weigh an amount of the same protein as the free enzyme (e.g., if the free enzyme protein content is 5 mg, the immobilized enzyme protein content should also be 5 mg) and resuspend it in 1 mL of sodium phosphate buffer (200 mM, pH 7.0). Incubate at 35℃ and 800 rpm in a metal bath for 10 min. After incubation, add the substrate and continue the reaction for 10 min. After the reaction is completed, add a trace amount of 6 M hydrochloric acid solution to terminate the reaction. Centrifuge at 12000 rpm for 1 min, collect the supernatant, dilute it 10 times, and perform liquid chromatography analysis.

[0086] The substrate and added amount of 0.1 M 1-cyanocyclohexylacetonitrile in the assay of free enzyme or immobilized enzyme activity R, S -O-chlorophenylglycine methyl ester, N -phenylacetyl-( R,S )-o-chlorophenylglycine, 1-methoxy-2-propanone, or 0.1 g castor meal powder; when the substrate is 1-methoxy-2-propanone, add isopropylamine as a co-substrate and pyridoxal phosphate (PLP) as a coenzyme. The molar ratio of 1-methoxy-2-propanone to isopropylamine is 1:1.5, and the final concentration of PLP is 1 mM.

[0087] Enzyme activity recovery rate calculation: The enzyme activity (U) of free enzyme was detected using HPLC. f The enzyme activity (U) of immobilized enzymes containing equal mass of protein catalyzing reactions with the same type and concentration of substrates was measured. I Calculate U I / U f The value represents the enzyme activity recovery rate of the immobilized enzyme.

[0088] Example 4: Construction of MOF-immobilized enzymes

[0089] (1) Construction of AcN-T2@ZIF-8

[0090] Natural positively charged ZIF-8 material was dried, ground, and dispersed in PBS buffer (pH 7.0). Crude enzyme solution of mutant AcN-T2 was added at a protein to ZIF-8 mass ratio of 1:25. The mixture was magnetically stirred at 4°C for 7 h to achieve directional electrostatic adsorption and immobilization. After centrifugation at 6000 rpm for 5 min, washing with ultrapure water, and drying at 25°C, immobilized AcN-T2 nitrile hydrolase was obtained, denoted as AcN-T2@ZIF-8, with an enzyme loading of 51.2 mg / g and an enzyme activity recovery rate of 85%.

[0091] (2) Construction of BS-M2@MIL-01

[0092] Naturally negatively charged MIL-101(Cr) material was dried, ground, and dispersed in PBS buffer (pH 8.0). Crude enzyme solution of mutant BS-M2 was added at a protein to MIL-101(Cr) mass ratio of 1:22. The mixture was magnetically stirred at 4°C for 6.5 h to achieve directional electrostatic adsorption and immobilization. After centrifugation at 6000 rpm for 5 min, washing with ultrapure water, and drying at 25°C, immobilized BS-M2 esterase was obtained, denoted as BS-M2@MIL-01, with an enzyme loading of 52.3 mg / g and an enzyme activity recovery rate of 86%.

[0093] (3) Construction of SM-M2@ZIF-8

[0094] Natural positively charged ZIF-8 material was dried, ground, and dispersed in PBS buffer (pH 7.0). Crude enzyme solution of mutant SM-M2 was added at a protein to ZIF-8 mass ratio of 1:24. The mixture was magnetically stirred at 4°C for 7 h to achieve directional electrostatic adsorption and immobilization. After centrifugation at 6000 rpm for 5 min, washing with ultrapure water, and drying at 25°C, immobilized SM-M2 acylase was obtained, denoted as SM-M2@ZIF-8, with an enzyme loading of 53.6 mg / g and an enzyme activity recovery rate of 93%.

[0095] (4) Construction of BM-M2@MIL-101

[0096] Naturally negatively charged MIL-101 material was dried, ground, and dispersed in PBS buffer (pH 7.8). Crude enzyme solution of mutant BM-M2 was added at a protein to MIL-101 mass ratio of 1:22. The mixture was magnetically stirred at 4°C for 6.5 h to achieve directional electrostatic adsorption and immobilization. After centrifugation at 6000 rpm for 5 min, washing with ultrapure water, and drying at 25°C, immobilized transaminase BM-M2 was obtained, denoted as BM-M2@MIL-101, with an enzyme loading of 54.2 mg / g and an enzyme activity recovery rate of 89%.

[0097] (5) Construction of NHasePs-M2@ZIF-8

[0098] Natural positively charged ZIF-8 material was dried, ground, and dispersed in PBS buffer (pH 7.5). Crude enzyme solution of mutant NHasePs-M2 was added at a mass ratio of enzyme to ZIF-8 of 1:25. The mixture was magnetically stirred at 4°C for 7 h to achieve directional electrostatic adsorption and immobilization. After centrifugation at 6000 rpm for 5 min, washing with ultrapure water, and drying at 25°C, immobilized nitrile hydratase NHasePs-M2 was obtained, denoted as NHasePs-M2@ZIF-8, with an enzyme loading of 54.5 mg / g and an enzyme activity recovery rate of 82%.

[0099] Example 5: AcN-T2@ZIF-8 catalytic production of 1-cyanocyclohexylacetic acid

[0100] PBS buffer (pH 7.0) and 500 mM substrate 1-cyanocyclohexylacetonitrile (1-CN) were added to a 100 mL reaction system. After stirring and dissolving, 0.25 g of AcN-T2@MOFs were added. The reaction was carried out at 35 °C and 600 r / min for 5 h. Real-time HPLC monitoring during the reaction showed that the conversion rate of 1-CN reached 98.1%, and the yield of the target product 1-cyanocyclohexylacetic acid (1-CA) was 97.5%. Figure 2 The reaction process diagram for the catalytic conversion of the target product 1-CA by AcN-T2@MOFs is shown. After the reaction, the immobilized enzyme was recovered by centrifugation. The recovered immobilized enzyme was reused 10 times, and the 1-CN conversion rate remained at around 60%.

[0101] Example 6: BS-M2@MIL-01 catalytic production ( S )-o-chlorophenylglycine

[0102] Add PBS buffer (pH 8.0) and substrate to a final concentration of 200 mM to a 50 mL reaction system. R,S 0.15 g of BS-M2@MIL-01 was added after stirring and dissolving methyl 1-o-chlorophenylglycine and reacted at 35℃ and 600 r / min for 8 h. Real-time HPLC analysis showed that the substrate conversion rate reached 48.2%. Figure 3 The target product was generated by BS-M2@MIL-01 catalysis. S The reaction process of 1,4-chlorophenylglycine is shown in the diagram. After the reaction, the immobilized enzyme was recovered by centrifugation. The recovered immobilized enzyme was reused 10 times, and the substrate conversion rate remained at 32.5%.

[0103] Example 7: SM-M2@ZIF-8 catalytic generation ( S )-o-chlorophenylglycine

[0104] Add PBS buffer (pH 7.0) and substrate N-phenylacetyl-(N ... R,S )-o-chlorophenylglycine was dissolved by stirring and then added to 0.35 g of SM-M2@ZIF-8. The reaction was carried out at 35℃ and 600 r / min for 24 h. Real-time HPLC detection showed that the substrate resolution conversion rate reached 45.8%. Figure 4 To catalyze the generation of (SM-M2@ZIF-8) S The reaction process of 1-o-chlorophenylglycine is shown in the diagram. After the reaction, the immobilized enzyme was recovered by centrifugation. The recovered immobilized enzyme was reused 12 times, and the substrate conversion rate remained above 33%.

[0105] Example 8: BM-M2@MIL-101 catalytic generation of (S)-(+)-1-methoxy-2-propane

[0106] PBS buffer (pH 7.8), 1-methoxy-2-propanone (final concentration 600 mM), 900 mM isopropylamine (amino donor), and 1 mM PLP were added to a 100 mL reaction system. After stirring and dissolving, 0.32 g of BM-M2@MIL-101 was added. The reaction was carried out at 30 °C and 800 r / min for 24 h. Real-time HPLC monitoring showed that the substrate conversion rate reached 99.7%. Figure 5 BM-M2@MIL-101 catalyzed generation ( S The reaction process of 1-(+)-1-methoxy-2-propane is shown in the diagram. After the reaction, the immobilized enzyme was recovered by centrifugation. The recovered immobilized enzyme was reused 15 times, and the substrate conversion rate remained above 65%.

[0107] Example 9: Degradation of ricinine in castor meal by NHasePs-M2@ZIF-8

[0108] PBS buffer (pH 7.5) and 50 g of castor bean cake powder that has been dried and pulverized at 50℃ were added to a 50 mL reaction system. After stirring to form a slurry, 0.5 g of NHasePs-M2@ZIF-8 was added. The reaction was carried out at 37℃ and 300 r / min for 12 h. Real-time HPLC detection showed that the initial ricin content was 3.1 mg / g, and the residual amount after the reaction was reduced to 0.28 mg / g, with a degradation rate of 91.0%. Figure 6 The reaction process diagram shows the degradation of ricinole in castor bean meal by immobilized nitrile hydratase. After the reaction, the reaction solution was filtered, and the filter cake was dried to obtain non-toxic crude castor bean meal.

Claims

1. A surface charge-modified enzyme mutant, characterized in that, The enzyme mutant is obtained by mutating two amino acids adjacent to the enzyme active center to change the local surface charge.

2. The enzyme mutant of claim 1, wherein The amino acid sequence of the enzyme mutant is shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.

10.

3. A MOFs immobilized enzyme of the enzyme mutant of claim 1, characterized in that, The immobilized enzyme is prepared by using MOFs as the carrier, using the crude enzyme solution extracted from the induced culture of the engineering bacteria expressing the enzyme mutant as the active component, and under the magnetic stirring at 4℃.

4. The MOFs-immobilized enzyme according to claim 3, wherein, The MOFs include MIL-101 (Cr) with natural negative electricity or ZIF-8 with natural positive electricity.

5. The MOFs-immobilized enzyme according to claim 3, wherein, The preparation method of the MOFs immobilized enzyme is as follows: the MOFs powder is added into a buffer solution with pH 2-8, the crude enzyme solution of the mutant enzyme is added, the reaction is stirred at 4℃ for 6-8 h to ensure the full contact between the enzyme and the carrier; after the reaction, the immobilized enzyme and the unfixed enzyme protein are separated by centrifugation at 6000 rpm for 5 min; finally, the precipitate is washed with ultrapure water and dried at 25℃ to obtain the immobilized enzyme; the protein content of the mutant enzyme is 1:22-25 to the mass of the MOFs; the buffer solution is used in an amount of 1-5 mL / mg to the mass of the carrier.

6. The use of the MOFs immobilized enzyme of claim 3 in the preparation of compound intermediates.

7. Use according to claim 6, wherein The compounds intermediates include gabapentin intermediates 1-cyanocyclohexylacetic acid, clopidogrel key intermediates ( S )-o-chlorophenylglycine, chemical intermediates S )-1-methoxy-2-propylamine.

8. Use according to claim 7, wherein the compound is ###0002### The application method is: taking MOFs immobilized enzyme as a catalyst, adding a substrate, taking purified water or a pH 7-8 sodium phosphate buffer as a reaction medium to form a reaction system, reacting under the condition of 30-35 DEG C and 600-800 rpm, obtaining a reaction liquid containing a product, separating and purifying the reaction liquid, recycling the catalyst for repeated use, and obtaining the intermediate; the substrate includes 1-cyanocyclohexylacetonitrile, R,S - o-chlorophenylglycine methyl ester, N - phenylacetyl-( R, S )-o-chlorophenylglycine, 1-methoxy-2-propanone; the catalyst is added in a final concentration of 1-5 g / L; the substrate is added in a final concentration of 200-1000 mM; when the substrate is 1-methoxy-2-propanone, isopropylamine is added, the molar concentration ratio of 1-methoxy-2-propanone to isopropylamine is 1:1.5, coenzyme pyridoxal phosphate is added, and the final concentration of the added pyridoxal phosphate is 1 mM.

9. The use of the MOFs immobilized enzyme of claim 3 in the degradation of gossypol in castor meal.

10. The use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The use is carried out according to the following steps: using the MOFs immobilized enzyme as the catalyst and the castor meal as the substrate, adding 200 mM, pH 7.5 sodium phosphate buffer to form a reaction system, and culturing at 37℃ and 300 rpm to achieve the degradation of gossypol; the mass ratio of the catalyst to the substrate is 1:80-100, and the amount of the buffer added to the castor meal is 100-1000 mL / kg.

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