WO2019157921A1 - 腈水解酶突变体及其应用 - Google Patents

腈水解酶突变体及其应用 Download PDF

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WO2019157921A1
WO2019157921A1 PCT/CN2019/072894 CN2019072894W WO2019157921A1 WO 2019157921 A1 WO2019157921 A1 WO 2019157921A1 CN 2019072894 W CN2019072894 W CN 2019072894W WO 2019157921 A1 WO2019157921 A1 WO 2019157921A1
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nitrilase
mutant
buffer
final concentration
seq
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PCT/CN2019/072894
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薛亚平
郑裕国
徐喆
柳志强
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浙江工业大学
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Priority to US16/619,344 priority Critical patent/US11001823B2/en
Publication of WO2019157921A1 publication Critical patent/WO2019157921A1/zh
Priority to US17/165,319 priority patent/US11525131B2/en
Priority to US17/165,153 priority patent/US11535839B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/002—Nitriles (-CN)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)

Definitions

  • the present invention relates to a mutant of a nitrilase derived from an uncultured microorganism and its use in the synthesis of an antiepileptic drug intermediate 1-cyanocyclohexylacetic acid.
  • the gabapentin chemical name is 1-aminomethyl-1-cyclohexaneacetic acid. Developed by Warner-Lambert, USA, it was first marketed in the UK in May 1993, and was approved by the FDA in the United States in 1994. It was later used in epilepsy treatment in many countries around the world. In 1996, Warner-Lambert began to expand. Indications for gabapentin indications, in 2002, the FDA approved its use in the treatment of neuropathic pain medications. In addition to being used alone in the treatment of general epilepsy, gabapentin is also used as a superimposed therapeutic for refractory epilepsy. It has the advantages of good tolerance and mild side effects, and is expected to promote the development of the world epilepsy drug market. one. At present, the patent of gabapentin has expired, and countries around the world have carried out research on this product. The demand for bulk drugs is huge and the market prospect is broad.
  • 1-Cyanocyclohexaneacetic acid is a key intermediate for the synthesis of a new generation of anti-epileptic drug Gabapentin.
  • the market prospect is very broad.
  • the synthesis of gabapentin including its key intermediate 1-cyanocyclohexyl acetic acid all adopts chemical synthesis technology, and there are problems of large discharge of three wastes, serious environmental pollution and high cost of treatment of three wastes in the production process.
  • Nitrilease EC (3.5.5.1) is an enzyme that hydrolyzes nitriles (containing -CN) to the corresponding carboxylic acid.
  • the cyanohydrolysis enzyme has a mild cyanolysis reaction condition, high reaction efficiency, high regioselectivity and stereoselectivity, mild reaction conditions, low environmental pollution and low cost, and is an environmentally friendly green synthesis method. In line with the requirements of the atomic economy, it has important practical significance for energy conservation and emission reduction and building a harmonious society. Due to these excellent properties of nitrilase, it has become a catalyst for industrial development. There are many examples of successful nitrilases in industrial applications.
  • the product (R)-mandelic acid of BASF in Germany is firstly reacted with benzaldehyde and hydrocyanic acid to form racemic mandelonitrile, and then the appropriate reaction conditions are selected to pass the nitrile.
  • Hydrodynamic enzyme-catalyzed dynamic kinetic resolution can be quantitatively converted to (R)-mandelic acid.
  • DuPont has developed an industrial process to convert 2-methylglutaronitrile (MGN, a by-product from the manufacture of nylon-66 from adiponitrile) to 1,5-dimethyl-2-piperidone (1,5-DMPD). 1,5-Dimethyl-2-piperidone has desirable properties in electronics, coatings and solvent applications.
  • MGN was first hydrolyzed to 4-cyanovaleric acid (4-CPA) ammonium salt by immobilized nitrilase-containing microbial cell catalyst (Acidovorax facilis 72W), the selectivity of hydrolysis reaction was greater than 98%, and the conversion rate was 100%. One-half of the ammonium cyanocarboxylate can be obtained, yielding 1-2% of the only reaction by-product, 2-methylglutarate diammonium salt. Compared to a chemical process that directly converts MGN hydrogenation to a mixture of 1,3-DPMD and 1,5-DMPD, the chemical-enzymatic process produces high yields, generates less waste, and produces a single lactam isomerization. body. In addition, many nitrilase enzymes have been developed and used in the synthesis of a variety of pharmaceutical intermediates and fine chemicals.
  • nitrilases are generally less thermally stable, which hinders their industrial applications.
  • Increasing the thermal stability of the enzyme can be achieved by chemical modification or molecular modification of the enzyme. Since the crystal structure of the nitrilase has been reported less, the modification of the stability of the nitrilase has rarely been reported.
  • the nitrilase cloned from Acidovorax facilis CCTCC NO: M 209044 has been overexpressed in Escherichia coli BL21 (DE3) and molecularly engineered to exhibit the substrate 1-cyanocyclohexylacetonitrile.
  • a higher catalytic activity is available to catalyze the production of gabapentin intermediate 1-cyanocyclohexyl acetic acid by 1-cyanocyclohexylacetonitrile (Catalysis Communications, 2015, 66, 121-125).
  • the biocatalyst also has problems of poor temperature stability, low catalytic efficiency at high temperature, and long reaction time.
  • nitrilase derived from Acidovorax facilis CCTCC NO:M 209044 was modified by error-prone PCR and site-directed mutagenesis to increase the temperature stability at 45 °C by 14 times.
  • the use of molecular modification technology to improve the temperature stability of nitrilase has important reference value.
  • the present invention provides a plurality of nitrilase mutant proteins and synthesis in 1-cyanocyclohexyl acetic acid for the problem of poor temperature stability derived from the uncultured microorganism nitrilase LNIT5 (GenBank Accession no: AAR97494.1).
  • the application includes a recombinant vector containing the gene, and a recombinant genetic engineering strain transformed by the recombinant vector.
  • the present invention provides a nitrilase mutant obtained by mutating the 201st position of the amino acid sequence shown in SEQ ID No. 2 or replacing the amino acid sequence of 324-381.
  • the mutant is one of the following: (1) mutating the leucine at position 201 of the amino acid sequence shown in SEQ ID No. 2 to phenylalanine, and the amino acid sequence of the mutant is as shown in SEQ ID No.
  • the nucleotide sequence of SEQ ID No. 3 (2) replaces amino acids 324-381 of the amino acid sequence shown in SEQ ID No. 2 with a hydrolyzed acid derived from Acidovorax facilis CCTCC NO: M 209044.
  • the amino acids 324-371 of the enzyme (amino acid sequence 324-371 is shown in SEQ ID No. 8, nucleotide sequence SEQ ID No. 7), the mutant amino acid sequence is shown in SEQ ID No. 6, nucleus
  • the nucleotide sequence is SEQ ID No. 5.
  • the invention utilizes the NCBI database to screen and obtain the gene derived from the uncultured microorganism nitrilase (GenBank Accession no: AAR97494.1), in order to realize its soluble expression in prokaryotic organisms such as Escherichia coli, through genetic engineering routine operation, to The synthetic method obtained the nucleotide sequence SEQ ID No. 1 of the nitrilase corresponding to the amino acid sequence shown in SEQ ID No. 2.
  • the amino acid sequence shown in SEQ ID No. 2 was mutated by site-directed mutagenesis.
  • PCR amplification was performed using primers, and site 201 was subjected to site-directed mutagenesis to obtain an expression vector pET-28b containing the nucleotide sequence of the nitrilase mutant. After (+), it was transferred to an E. coli host to induce expression to obtain a mutant with improved temperature stability, thereby obtaining a mutant protein LNIT5-L201F capable of efficiently catalyzing the selective hydrolysis of a dinitrile compound to a monocyanocarboxylic acid compound.
  • Nucleotide sequence SEQ ID No. 3 Nucleotide sequence SEQ ID No. 3).
  • primers were first designed to amplify A. facilis CTCCC NO: M 209044 (GenBank Accession no. KJ001820) nitrilase from the 324-371.
  • the nucleotide sequence corresponding to the amino acid SEQ ID No. 7
  • primers were designed, and the homologous arm containing the homologous arm and the gene derived from the uncultured microorganism nitrilase (GenBank Accession no: AAR97494.1) were amplified by PCR.
  • the present invention also relates to a gene encoding the nitrilase mutant, a recombinant vector constructed from the coding gene, and a recombinant genetically engineered bacterium obtained by transforming the host cell into the recombinant vector.
  • the host cell may be various conventional host cells in the art, and E. coli BL21 (DE3) is preferred in the present invention.
  • the synthesized nitrilase gene was ligated to the expression vector pET-28b(+) by restriction enzyme ligation to construct a recombinant expression vector pET-28b(+)-LNIT5.
  • the present invention also provides a recombinant engineered strain comprising the above nitrilase gene LNIT5, preferably Escherichia coli BL21 (DE3).
  • LNIT5 preferably Escherichia coli BL21 (DE3).
  • the recombinant expression vector pET-28b(+)-LNIT5 described above was transformed into the host cell E. coli BL21 (DE3) to obtain the recombinant strain.
  • the invention also provides the use of the nitrilase mutant in catalyzing the preparation of a monocyanocarboxylic acid compound by using a nitonitrile compound, wherein the application is obtained by fermentation culture of a genetically engineered strain containing a nitrilase-containing mutant.
  • the sodium buffer is a reaction medium, and after the reaction is completed in a constant temperature water bath at 45° C., the reaction solution is separated and purified to obtain 1-cyanocyclohexyl acetic acid; the final concentration of the substrate is 100 to 1300 mM, preferably 1000 mM, based on the volume of the buffer.
  • the amount of the catalyst is from 10 to 100 g/L of the buffer, preferably 50 g/L, based on the weight of the wet cells.
  • the catalyst is prepared according to one of the following methods: (1) inoculating a RN-containing hydrolase-containing genetically engineered strain into an LB medium, incubating at 37 ° C for 10-12 hours, and inoculating at a volume concentration of 2%.
  • the LB medium containing the final concentration of 50 mg/L kanamycin was expanded and cultured at 37 ° C until the OD 600 of the culture solution was between 0.6 and 0.8, and the final concentration was 0.1 mM isopropyl- ⁇ -D-sulfur.
  • step (1) wet cells with final concentration 300 mM NaCl pH 8.0, 50 mM NaH 2 PO 4 buffer was resuspended, sonicated (400 W, 15 min, 1 s break 1 s pause), the crushed product was centrifuged (12000 ⁇ g, 20 min) and the supernatant was taken as the crude enzyme solution;
  • the crude enzyme solution was passed through a Ni-NTA column flushed with equilibration buffer at a flow rate of 1 mL/min, and the weakly adsorbed heteroprotein was eluted with an elution buffer at a flow rate of 2 mL/min; and eluted with protein elution buffer and
  • the target protein was collected at a flow rate of 2 mL/min; finally, the collected target protein was treated with a mass concentration of
  • the equilibration buffer to a final concentration having pH 8.0,50mM NaH 2 PO 4 300mM NaCl in buffer solution; the final concentration of the elution containing 300mM NaCl and 50mM imidazole pH buffer 8.0, 50 mM NaH 2 PO 4 buffer; the protein elution buffer was pH 8.0, 50 mM NaH 2 PO 4 buffer containing a final concentration of 300 mM NaCl and 250 mM imidazole.
  • the nitrilase mutant of the present invention may be a recombinant expression transformant (ie, a wet cell, preferably Escherichia coli E. coli BL21 (DE3)) containing the nitrilase mutant gene, or may be unpurified.
  • a recombinant expression transformant ie, a wet cell, preferably Escherichia coli E. coli BL21 (DE3)
  • the crude enzyme solution, or the purified pure enzyme can be immobilized if necessary.
  • Luria-Bertani (LB) liquid medium final concentration composition 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, the solvent is water, the pH is natural.
  • LB solid medium final concentration composition 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, agar 1.5%, the solvent is water, the pH is natural.
  • the beneficial effects of the invention are mainly embodied in: the invention is modified by protein molecule, the thermal stability of the nitrilase LNIT5 pure enzyme is up to 4.5 times, and the recombinant large intestine containing the nitrilase mutant is utilized.
  • Bacillus hydrolyzed 1-cyanocyclohexylacetonitrile at high temperature (45 ° C) the product tolerance was improved, NIT5-L201F activity was increased by 20%, and the mutant NITLNIT5-AcN increased the conversion rate at 750 mM substrate concentration.
  • complete conversion of 750 mM 1-cyanocyclohexylacetonitrile can be completed in 8 hours. Therefore, the mutant obtained by the present invention has a good application prospect in the high-efficiency catalysis of the synthesis of gabapentin intermediate 1-cyanocyclohexyl acetic acid by 1-cyanocyclohexylacetonitrile.
  • Figure 4 is a graph comparing the time of transformation of recombinant E. coli resting cells containing nitrilase mutants to 400 mM 1-cyanocyclohexylacetonitrile.
  • Figure 5 is a graph showing the comparison time of 750 mM 1-cyanocyclohexylacetonitrile in recombinant E. coli resting cells containing nitrilase mutants.
  • Figure 6 Time plot of transformation of 1.0 M 1-cyanocyclohexylacetonitrile in recombinant E. coli resting cells containing nitrilase mutants.
  • Figure 7 High performance liquid chromatogram of 1-cyanocyclohexyl acetic acid.
  • BLAST was carried out using a nitrilase derived from A. facilis CTCCC NO: M 209044 as a template, and a nitrilase gene was screened from the results (GenBank Accession no: AAR97494.1) .
  • the nitrilase is open sourced to an uncultured microorganism which is 76% similar to the CTCCC NO:M 209044 nitrilase derived from A. facilis.
  • the nitrilase gene LNIT5 is represented by SEQ ID No. 2, and the amino acid sequence encoding the enzyme is as shown in SEQ ID No. 1. Show.
  • Example 2 Construction of recombinant expression vector pET-28b(+)-LNIT5 and construction of recombinant engineering bacteria
  • the recombinant expression vector pET-28b(+)-LNIT5 containing the nitrilase gene LNIT5 was synthesized by a synthetic method by a conventional method of genetic engineering.
  • the constructed expression vector pET-28b(+)-LNIT5 was transformed into E. coli BL21 (DE3) recipient strain and plated on LB agar plates containing kanamycin (final concentration 50 ⁇ g/mL), 37 Incubate overnight at °C. The colonies were randomly picked on the plate, and the plasmid was extracted and identified by agarose gel electrophoresis to obtain recombinant genetic engineering bacteria E. coli BL21(DE3)/pET-28b(+)-LNIT5.
  • Site-directed mutagenesis was carried out by whole plasmid amplification using the expression plasmid pET-28b(+)-LNIT5 as a template.
  • the PCR system 50 ⁇ L was: template 0.5-20 ng, primers L201F-f and L201F-r (see Table 1 for each sequence) 10-15 pmol, 5 ⁇ PrimeSTAR Buffer (Mg 2+ plus), 0.2 mM dNTP, 1.25 U PrimeSTAR HS DNA Polymerase.
  • PCR conditions (1) pre-denaturation at 98 ° C for 3 min; (2) denaturation at 98 ° C for 10 s; (3) annealing at 60 ° C for 5 s; (4) extension at 72 ° C for 6.5 min, steps (2) to (4) for a total of 30 cycles; 5) The last 72 ° C extension for 5 min, 4 ° C preservation.
  • the PCR product was verified by agarose gel electrophoresis, digested with DpnI, introduced into host strain E. coli BL21 (DE3), and applied to an LB plate containing 50 ⁇ g/mL kanamycin to obtain a monoclonal antibody.
  • the picked monoclonal plasmid was verified by gene sequencing of Beijing Qingke Biotechnology Co., Ltd. to obtain the mutant LNIT5-L201F, the amino acid sequence of which is shown in SEQ ID No. 4, and the nucleotide sequence is SEQ ID. No.3 is shown.
  • the recombinant expression plasmid pET-28b(+)-AcN containing the nitrilase AcN derived from A. facilis CTCCC NO: M 209044 was used as a template to obtain homologous arm and nitrile hydrolysis by PCR amplification.
  • the nucleotide sequence corresponding to the amino acid at positions C-324-371 of the enzyme AcN is represented by SEQ ID No. 8, nucleotide sequence SEQ ID No. 7).
  • the PCR system (50 ⁇ L) was: template 0.5-20 ng, primers If and Ir each 10-15 pmol, 5 ⁇ PrimeSTAR Buffer (Mg 2+ plus), 0.2 mM dNTP, 1.25 U PrimeSTAR HS DNA Polymerase.
  • the PCR conditions were 1) predenaturation at 98 °C for 3 min; (2) denaturation at 98 °C for 10 s; (3) annealing at 60 °C for 5 s; (4) extension at 72 °C for 10 s, and steps (2) to (4) for a total of 30 cycles; The last 72 ° C extension for 5 min, 4 ° C preservation.
  • PCR product was separated by agarose gel electrophoresis, and a DNA fragment of about 150 bp was recovered for use.
  • pET-28b(+)-LNIT5 as a template, pET-28b(+) containing the nucleotide sequence corresponding to the N-terminal 1-323 amino acid in the homologous arm and the nitrilase LNIT5 was amplified by PCR.
  • Linear plasmid sequence The PCR system (50 ⁇ L) was: template 0.5-20 ng, primers Pf and Pr 10-15 pmol each, 5 ⁇ PrimeSTAR Buffer (Mg 2+ plus), 0.2 mM dNTP, 1.25 U PrimeSTAR HS DNA Polymerase.
  • the PCR conditions were 1) predenaturation at 98 °C for 3 min; (2) denaturation at 98 °C for 10 s; (3) annealing at 60 °C for 5 s; (4) extension at 72 °C for 6.5 min, and steps (2) to (4) for a total of 30 cycles; 5) The last 72 ° C extension for 5 min, 4 ° C preservation.
  • the PCR product was verified by agarose gel electrophoresis, digested with restriction endonuclease DpnI, and the desired fragment was obtained by PCR purification kit. Finally, use II One Step Cloning Kit (Nanjing Nuoweizan Biotechnology Co., Ltd.) achieves homologous recombination.
  • the expression plasmid containing the fusion protein was introduced into a host strain E. coli BL21 (DE3), and applied to an LB plate containing 50 ⁇ g/mL kanamycin to obtain a monoclonal antibody.
  • the picked monoclonal plasmid was verified by gene sequencing of Beijing Qingke Biotechnology Co., Ltd. to obtain the fusion protein NIT LNIT5-AcN , the amino acid sequence of which is shown in SEQ ID No. 6, and the nucleotide sequence is SEQ. ID No. 5 is shown.
  • Example 2 and Example 3 The transformants obtained in Example 2 and Example 3 were E. coli BL21(DE3)/pET-28b(+)-LNIT5, E.coli BL21(DE3)/pET-28b(+)-LNIT5-L201F and E. Coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN , inoculated into LB medium, cultured at 37 °C for 10-12 hours, inoculated with 2% by volume to contain kanamycin (final concentration 50 mg) /L) LB medium was expanded (37 ° C).
  • the OD 600 of the culture solution was between 0.6 and 0.8, and isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and culture was induced at 28 ° C for 10 hours.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • Example 4 To the wet cells collected in Example 4, 50 mM NaH 2 PO 4 , 300 mM NaCl buffer (pH 8.0) was added, and the cells were resuspended and sonicated (400 W, 15 min, 1 s break for 1 s). The crushed product was centrifuged (12,000 ⁇ g, 20 min), and the supernatant was taken as a crude enzyme solution to prepare for separation and purification.
  • the weakly adsorbed heteroprotein was eluted using an elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 50 mM imidazole, pH 8.0) at a flow rate of 2 mL/min.
  • an elution buffer 50 mM NaH 2 PO 4 , 300 mM NaCl, 50 mM imidazole, pH 8.0
  • the protein of interest was eluted and collected using a protein elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8.0) at a flow rate of 2 mL/min.
  • a protein elution buffer 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8.0
  • the collected enzyme solution was dialysis bag (Economical Biotech Membrane, 14KD, 34mm Width, purchased from Bioengineering Biotechnology (Shanghai) Co., Ltd.), and the dialysate was a 0.9% sodium chloride aqueous solution. The retentate was taken. Purified protein.
  • the purified protein was analyzed by SDS-PAGE.
  • Example 6 Determination of nitrilase activity
  • Nitrile Hydrolase Activity Detection Reaction System (10 mL): Buffer sodium dihydrogen phosphate-disodium hydrogen phosphate (100 mM, pH 7.0), 200 mM 1-cyanocyclohexylacetonitrile, 75 mg pure enzyme. The reaction solution was preheated at 45 ° C for 10 min and then reacted at 150 rpm for 10 min. 500 ⁇ L of the supernatant was sampled, and after 500 ⁇ L of 2 M HCl was added to terminate the reaction, the conversion of 1-cyanocyclohexyl acetic acid was measured by liquid chromatography (Shimadzu LC-16) external standard method.
  • Example 7 Determination of temperature stability at 45 ° C in nitrilase mutants
  • Example 5 The protein after purification in Example 5 was measured for thermal stability. A certain amount of protein was taken in a 50 mL sterile polypropylene centrifuge tube and stored in a constant temperature water bath at 45 °C. The protein was taken at different times and the viability of the protein was determined according to the method of Example 6. The relative activity of the protein at each time was calculated as the control when the activity of the protein was not preserved.
  • Example 8 Determination of Recombinant E. coli Activity Containing a Nitrile Hydrolase Mutant
  • Example 9 Determination of temperature stability of recombinant Escherichia coli containing nitrilase mutant at 45 ° C
  • the recombinant Escherichia coli BL21(DE3)/pET-28b(+)-LNIT5, E.coli BL21(DE3)/pET-28b(+)- containing the nitrilase mutant obtained in Example 4 was cultured.
  • LNIT5-L201F and E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN resting cells were configured with buffer sodium dihydrogen phosphate-disodium hydrogen phosphate (200 mM, pH 7.0) to a concentration of 20 g/L.
  • the bacterial suspension was stored in a constant temperature water bath at 45 °C.
  • the bacterial suspension was taken out at different times, and the viability of resting cells was measured in accordance with the method of Example 8.
  • the relative residual viability of resting cells at each time was calculated by taking the viability of resting cells as a control, and the results are shown in Table 2.
  • Example 10 Transformation of 400 mM 1-cyanocyclohexylacetonitrile using recombinant E. coli containing a nitrilase mutant
  • E. coli BL21(DE3)/pET-28b(+)-LNIT5 E. coli BL21(DE3)/pET-28b(+)-LNIT5-L201F and E.coli obtained by the method of Example 4, respectively.
  • E. coli BL21 (DE3) / pET-28b (+) - LNIT5 - L201F and E. coli BL21 ( DE3)/pET-28b(+)-NIT LNIT5-AcN can completely react the substrate within 6h, of which E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN is faster than E.coli BL21(DE3)/pET-28b(+)-LNIT5.
  • Example 11 Transformation of 750 mM 1-cyanocyclohexylacetonitrile using recombinant E. coli containing the nitrilase mutant NIT LNIT5-AcN
  • Example 12 Transformation of 1.0 M 1-cyanocyclohexylacetonitrile using recombinant E. coli containing the nitrilase mutant NIT LNIT5-AcN
  • Example 13 Transformation of 750 mM 1-cyanocyclohexylacetonitrile using immobilized cells

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Abstract

一种腈水解酶突变体及其应用，突变体是将SEQ ID No.2所示氨基酸序列的第201位氨基酸，或对第324-381位氨基酸序列进行一位或多位替换获得的。经过蛋白质分子改造，腈水解酶LNIT5纯酶在45℃下的热稳定性最高提高了4.5倍，且利用含有腈水解酶突变体的重组大肠杆菌在高温下(45℃)水解1-氰基环己基乙腈，产物得率得到了提高。因此，所获得的突变体在高效催化1-氰基环己基乙腈合成加巴喷丁中间体1-氰基环己基乙酸中具有良好的应用前景。

Description

腈水解酶突变体及其应用 技术领域
本发明涉及一种来源于未培养微生物的腈水解酶的突变体及其在抗癫痫药物中间体1-氰基环己基乙酸合成中的应用。
背景技术
加巴喷丁化学名为1-氨甲基-1-环己烷乙酸。由美国Warner-Lambert公司开发，于1993年5月首先在英国上市，1994年获得FDA批准在美国上市，后来陆续在全世界众多国家被用于癫痫病治疗，1996年，Warner-Lambert公司开始扩大加巴喷丁适应症的研究，2002年，FDA批准将其用于治疗神经病理性疼痛药。加巴喷丁除可单独用于治疗一般癫痛病外，还被作为难治性癫痫病的叠加治疗药，它具有耐受性良好，副作用轻微的优点，是人们期待的推动世界癫痫药物市场发展的药物之一。目前，加巴喷丁的专利已经到期，世界各国纷纷开展对于该产品的研究，原料药需求巨大，市场前景广阔。
1-氰基环己基乙酸(1-Cyanocyclohexaneacetic acid)是合成新一代抗癫痫药物加巴喷丁(Gabapentin)的关键中间体，市场前景非常广阔。目前，用于合成加巴喷丁包括其关键中间体1-氰基环己基乙酸全部采用化学合成技术，生产过程中存在三废排放量大，环境污染严重，三废处理成本高等问题。
腈水解酶(Nitrilase EC 3.5.5.1)是一种能将腈类物质(含有-CN)水解为相应羧酸的酶。通过腈水解酶实现氰基水解反应条件温和，反应效率高，而且具有比较高的区域选择性和立体选择性，反应条件温和，环境污染小，成本低，是一种环境友好的绿色合成方法，符合原子经济的要求，对节能减排及建设和谐社会具有重要的现实意义。由于腈水解酶的这些优异的特性，使其成为工业上极具发展潜力的催化剂。目前工业化应用中比较成功的腈水解酶的例子很多，德国BASF公司的产品(R)-扁桃酸，首先由苯甲醛和氢氰酸反应生成消旋扁桃腈，再选择合适的反应条件，通过腈水解酶催化的动态动力学拆分，可以定量地转化为(R)-扁桃酸。杜邦公司开发了一套工业化生产过程，将2-甲基戊二腈(MGN，己二腈制造尼龙-66过程中产生的副产物)转化为1,5-二甲基-2-哌啶酮(1,5-DMPD)。1,5-二甲基-2-哌啶酮在电子学、涂层及溶剂应用中具有令人满意的特性。MGN首先用固定化的含腈水解酶的微生物细胞催化剂(Acidovorax facilis 72W)水解为4-氰基戊酸(4-CPA)铵盐，水解反应的选择性大于98％，转化率为100％，能得到二分之一氰基羧酸铵盐，产生1～2％的唯一反应副产物2-甲基戊二酸二铵盐。与一个由MGN加氢直接转化为1,3-DPMD和1,5-DMPD混合物的化学工艺相比，化学-酶法生产工艺产量高、产生的浪费较少，并生成单一的内酰胺异构体。此外，许多腈水解酶已经被开发并用于多种药物中间体和精细化学品的合成。
但是，天然的腈水解酶热稳定性普遍较差，这阻碍了其工业应用。提高酶的热稳定性，可通过对酶进行化学修饰或分子改造而实现。由于腈水解酶的晶体结构报道较少，对于腈水解酶稳定性的改造也罕有报道。
从敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044克隆的腈水解酶已经在大肠杆菌(Escherichia coli)BL21(DE3)中过量表达，通过分子改造，对底物1-氰基环己基乙腈表现出了较高的催化活力，能够催化1-氰基环己基乙腈生产加巴喷丁的中间体1-氰基环己基乙酸(Catalysis Communications,2015，66,121-125)。但是，该生物催化剂还存在温度稳定性较差，高温下催化反应效率较低，反应时间较长等问题。后续的研究中，利用易错PCR及定点突变技术改造来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044的腈水解酶，使其在45℃下的温度稳定性提高了14倍，对于利用分子改造技术提高腈水解酶温度稳定性具有重要的参考价值。
发明内容
本发明要针对来源于未培养微生物腈水解酶LNIT5(GenBank Accession no:AAR97494.1)温度稳定性较差的问题，提供了多个腈水解酶突变体蛋白及在1-氰基环己基乙酸合成中的应用，包括含有该基因的重组载体，以及重组载体转化得到的重组基因工程菌。
本发明采用的技术方案是：
本发明提供一种腈水解酶突变体，所述突变体是将SEQ ID No.2所示氨基酸序列的第201位进行突变，或对第324-381位氨基酸序列中进行替换获得的。
进一步，优选所述突变体为下列之一：(1)将SEQ ID No.2所示氨基酸序列的第201位亮氨酸突变为苯丙氨酸，突变体氨基酸序列如SEQ ID No.4所示，核苷酸序列SEQ ID No.3；(2)将 SEQ ID No.2所示氨基酸序列第324-381位氨基酸替换为来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044腈水解酶的第324-371位氨基酸(第324-371位氨基酸序列如SEQ ID No.8所示，核苷酸序列SEQ ID No.7)，突变体氨基酸序列如SEQ ID No.6所示，核苷酸序列SEQ ID No.5。
本发明利用NCBI数据库，筛选得到来源于未培养微生物腈水解酶编码基因(GenBank Accession no:AAR97494.1)，为了实现其在大肠杆菌等原核生物中的可溶性表达，通过基因工程常规操作，以全合成的方法获得了对应于SEQ ID No.2所示氨基酸序的该腈水解酶的核苷酸序列SEQ ID No.1。利用定点突变技术对SEQ ID No.2所示氨基酸序进行突变，首先利用引物进行PCR扩增，对第201位进行定点突变，得到含有腈水解酶突变体核苷酸序列的表达载体pET-28b(+)后，转入大肠杆菌宿主，诱导表达后得到温度稳定性提高的突变体，从而获得能够高效催化双腈化合物区域选择性水解合成单氰基羧酸化合物的突变体蛋白LNIT5-L201F(核苷酸序列SEQ ID No.3)。
利用同源重组原理，首先设计引物，利用PCR扩增包含同源臂的来源于敏捷食酸菌(A.facilis)CCTCC NO:M 209044(GenBank Accession no.KJ001820)腈水解酶中第324-371位氨基酸所对应的核苷酸序列(SEQ ID No.7)；其次，设计引物，利用PCR扩增包含同源臂及来源于未培养微生物腈水解酶编码基因(GenBank Accession no:AAR97494.1)第1-323位氨基酸所对应的核苷酸序列的线性化载体序列；利用同源重组将两段核苷酸序列融合，得到含有腈水解酶融合体核苷酸序列的重组表达载体pET-28b(+)，转入大肠杆菌宿主，诱导表达后得到温度稳定性提高的突变体，从而获得能够高效催化双腈化合物区域选择性水解合成单氰基羧酸化合物的突变体蛋白NIT LNIT5-AcN(核苷酸序列SEQ ID No.5)。
本发明还涉及一种所述腈水解酶突变体的编码基因、由所述编码基因构建的重组载体以及由所述重组载体转化宿主细胞获得的重组基因工程菌。所述的宿主细胞可以是本领域的各种常规宿主细胞，本发明优选大肠杆菌E.coli BL21(DE3)。具体地，合成后的腈水解酶基因通过酶切连接的方法连接到表达载体pET-28b(+)上，构建成为重组表达载体pET-28b(+)-LNIT5。本发明还提供了包含上述腈水解酶基因LNIT5的重组工程菌株，优选为大肠杆菌BL21(DE3)。将上述的重组表达载体pET-28b(+)–LNIT5转化到宿主细胞大肠杆菌BL21(DE3)之中得到该重组菌株。
本发明还提供一种所述腈水解酶突变体在催化双腈化合物制备单氰基羧酸化合物中的应用，具体所述的应用以含腈水解酶突变体编码基因工程菌经发酵培养获得的湿菌体、湿菌体固定化细胞或者湿菌体超声破碎后提取的纯酶为催化剂，以1-氰基环己基乙腈为底物，以pH＝7.0、200M磷酸氢二钠-磷酸二氢钠缓冲液为反应介质，45℃恒温水浴反应完全后，将反应液分离纯化，获得1-氰基环己基乙酸；所述底物终浓度以缓冲液体积计为100～1300mM，优选1000mM，所述催化剂用量以湿菌体重量计为10～100g/L缓冲液，优选50g/L。
进一步，所述催化剂按如下方法之一制备：(1)将含腈水解酶突变体编码基因工程菌接种到LB培养基中，37℃培养10-12小时，按体积浓度2％的接种量接种至含终浓度50mg/L卡那霉素的LB培养基进行扩大培养，37℃培养至培养液的OD 600为0.6-0.8之间，加入终浓度为0.1mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，28℃诱导培养10小时，离心，收集菌体，用生理盐水清洗2次，得到湿菌体；(2)将步骤(1)湿菌体用含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液重悬，超声波破碎(400W,15min,1s破碎1s暂停)，破碎产物离心(12000×g,20min)后取上清液作为粗酶液；将粗酶液以1mL/min的流速通过经平衡缓冲液冲洗的Ni-NTA柱，用洗脱缓冲液洗脱弱吸附的杂蛋白，流速为2mL/min；再用蛋白洗脱缓冲液洗脱并收集目的蛋白，流速为2mL/min；最后将收集的目的蛋白以质量浓度0.9％氯化钠水溶液为透析液进行透析，取截留液即为纯酶；所述平衡缓冲液为含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液；所述洗脱缓冲液为含终浓度300mM NaCl和50mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液；所述蛋白洗脱缓冲液为含终浓度300mM NaCl和250mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液。
本发明所述的腈水解酶突变体可以是含有该腈水解酶突变体基因的重组表达转化体(即湿菌体，优选为大肠杆菌E.coli BL21(DE3))，也可以是未纯化的粗酶液，或是经过纯化后的纯酶，如果需要可以对其进行固定化后进行使用。
Luria-Bertani(LB)液体培养基终浓度组成：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，溶剂为水，pH值自然。
LB固体培养基质量终浓度组成：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，琼脂1.5％，溶剂为水，pH值自然。
与现有技术相比，本发明的有益效果主要体现在：本发明经过蛋白质分子改造，腈水解酶 LNIT5纯酶的热稳定性最高提高了4.5倍，且利用含有腈水解酶突变体的重组大肠杆菌在高温下(45℃)水解1-氰基环己基乙腈，产物耐受性得到提高，NIT5-L201F活力提高了20％，而突变体NITLNIT5-AcN在750mM底物浓度下，转化率提高了1倍，可以在8小时内完成750mM 1-氰基环己基乙腈的完全转化。因此，本发明所获得的突变体在高效催化1-氰基环己基乙腈合成加巴喷丁中间体1-氰基环己基乙酸中具有良好的应用前景。
附图说明
图1腈水解酶突变体纯酶活力的比较。
图2腈水解酶突变体在45℃下的热稳定性。
图3含有腈水解酶突变体的重组大肠杆菌静息细胞活力比较。
图4含有腈水解酶突变体的重组大肠杆菌静息细胞转化400mM 1-氰基环己基乙腈时间比较曲线图。
图5含有腈水解酶突变体的重组大肠杆菌静息细胞转化750mM 1-氰基环己基乙腈比较时间曲线图。
图6含有腈水解酶突变体的重组大肠杆菌静息细胞转化1.0M 1-氰基环己基乙腈时间曲线图。
图7 1-氰基环己基乙酸高效液相色谱图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：腈水解酶LNIT5的获得
根据蛋白质NCBI数据库，利用来源于敏捷食酸菌(A.facilis)CCTCC NO:M 209044的腈水解酶为模板进行BLAST，从结果中筛选得到一个腈水解酶基因(GenBank Accession no:AAR97494.1)。该腈水解酶开源于未培养微生物，其与来源于敏捷食酸菌(A.facilis)CCTCC NO:M 209044腈水解酶相似度为76％。根据该睛水解酶的氨基酸序列，并依据大肠杆菌偏好的密码子进行密码子优化，该段腈水解酶基因LNIT5如SEQ ID No.2所示，编码酶的氨基酸序列如SEQ ID No.1所示。
实施例2：重组表达载体pET-28b(+)-LNIT5的构建以及重组工程菌的构建
通过基因工程的常规操作以全合成的方法合成了含有腈水解酶基因LNIT5的重组表达载体pET-28b(+)-LNIT5。将构建的表达载体pET-28b(+)-LNIT5转化至E.coli BL21(DE3)受体菌中，涂布于含卡那霉素(终浓度为50μg/mL)的LB琼脂平板上，37℃培养过夜。于平板上随机挑取菌落，并抽提质粒进行琼脂糖凝胶电泳鉴定，获得重组基因工程菌E.coli BL21(DE3)/pET-28b(+)-LNIT5。
表1引物设计表
Figure PCTCN2019072894-appb-000001
实施例3：腈水解酶LNIT5突变体的构建
以表达质粒pET-28b(+)-LNIT5为模版，通过全质粒扩增进行定点突变。PCR体系(50μL)为：模版0.5-20ng，引物L201F-f及L201F-r(序列见表1)各10-15pmol，5×PrimeSTAR Buffer(Mg 2+plus)，0.2mM dNTP，1.25U PrimeSTAR HS DNA Polymerase。PCR条件(1)98℃预变性3min；(2)98℃变性10s；(3)60℃退火5s；(4)72℃延伸6.5min，步骤(2)～(4)共30个循环；(5)最后72℃延伸5min，4℃保存。PCR产物经过琼脂糖凝胶电泳验证，利用DpnI消化后导入宿主菌E.coli BL21(DE3)，涂布至含有50μg/mL卡那霉素的LB平板，得到单克隆。将挑取的单克隆抽提质粒，由北京擎科新业生物技术有限公司基因测序验证，得到突变体LNIT5-L201F，其氨基酸序列如SEQ ID No.4所示，核苷酸序列如SEQ ID No.3所示。
以含有来源于敏捷食酸菌(A.facilis)CCTCC NO:M 209044的腈水解酶AcN的重组表达质粒pET-28b(+)-AcN为模板，通过PCR扩增得到含有同源臂及腈水解酶AcN其C端324-371位氨基酸所对应的核苷酸序列(第324-371位氨基酸序列如SEQ ID No.8所示，核苷酸序列SEQ ID  No.7)。PCR体系(50μL)为：模板0.5-20ng，引物I-f及I-r各10-15pmol，5×PrimeSTAR Buffer(Mg 2+plus)，0.2mM dNTP，1.25U PrimeSTAR HS DNA Polymerase。PCR条件为1)98℃预变性3min；(2)98℃变性10s；(3)60℃退火5s；(4)72℃延伸10s，步骤(2)～(4)共30个循环；(5)最后72℃延伸5min，4℃保存。得到的PCR产物进行琼脂糖凝胶电泳分离，对150bp左右的DNA片段进行回收待用。以表达质粒pET-28b(+)-LNIT5为模版，通过PCR扩增得到含有同源臂以及腈水解酶LNIT5中N端1-323位氨基酸所对应的核苷酸序列的pET-28b(+)线性质粒序列。PCR体系(50μL)为：模板0.5-20ng，引物P-f及P-r各10-15pmol，5×PrimeSTAR Buffer(Mg 2+plus)，0.2mM dNTP，1.25U PrimeSTAR HS DNA Polymerase。PCR条件为1)98℃预变性3min；(2)98℃变性10s；(3)60℃退火5s；(4)72℃延伸6.5min，步骤(2)～(4)共30个循环；(5)最后72℃延伸5min，4℃保存。PCR产物经过琼脂糖凝胶电泳验证，使用限制性内切酶DpnI消化，利用PCR纯化试剂盒得到目的片段。最后，利用
Figure PCTCN2019072894-appb-000002
II One Step Cloning Kit(南京诺唯赞生物科技有限公司)实现同源重组。将该含有融合蛋白的表达质粒导入宿主菌E.coli BL21(DE3)，涂布至含有50μg/mL卡那霉素的LB平板，得到单克隆。将挑取的单克隆抽提质粒，由北京擎科新业生物技术有限公司基因测序验证，得到融合蛋白NIT LNIT5-AcN，其氨基酸序列如SEQ ID No.6所示，核苷酸序列如SEQ ID No.5所示。
实施例4：腈水解酶突变体的表达
实施例2和实施例3中得到的转化子E.coli BL21(DE3)/pET-28b(+)-LNIT5，E.coli BL21(DE3)/pET-28b(+)-LNIT5-L201F和E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN，接种到LB培养基中，37℃培养10-12小时，按体积接种量2％接种至含有卡那霉素(终浓度50mg/L)的LB培养基进行扩大培养(37℃)。待培养液的OD 600为0.6-0.8之间，加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)至终浓度为0.1mM，28℃诱导培养10小时。培养液离心收集菌体，用生理盐水清洗2次，得到湿菌体。
实施例5：腈水解酶突变体蛋白的纯化
(1)向实施例4中收集到的湿菌体中加入50mM NaH 2PO 4，300mM NaCl缓冲液(pH 8.0)，重悬菌体后超声波破碎(400W,15min,1s破碎1s暂停)。破碎产物离心(12000×g,20min)后取上清液作为粗酶液，准备分离纯化。
(2)预装好20mL Ni-NTA亲和层析柱后，使用平衡缓冲液(50mM NaH 2PO 4，300mM NaCl，pH 8.0)进行平衡，流速为2mL/min。
(3)清洗8-10个柱体积后将所得到的粗酶液以1mL/min的流速通过Ni-NTA柱，目的蛋白则挂载在层析柱上。上样后大量未吸附的杂蛋白不与树脂结合，将直接被除去。
(4)使用洗脱缓冲液(50mM NaH 2PO 4，300mM NaCl，50mM咪唑，pH 8.0)洗脱弱吸附的杂蛋白，流速为2mL/min。
(5)使用蛋白洗脱缓冲液(50mM NaH 2PO 4，300mM NaCl，250mM咪唑，pH 8.0)洗脱并收集目的蛋白，流速为2mL/min。
(6)收集的酶液使用透析袋(Economical Biotech Membrane，14KD，34mm Width，购自生工生物工程(上海)股份有限公司)，透析液为质量浓度0.9％氯化钠水溶液，取截留液即为纯化后的蛋白。
(7)通过SDS-PAGE分析纯化后的蛋白。
实施例6：腈水解酶活力的测定
对实施例5中纯化后的蛋白进行酶活的测定。腈水解酶活力检测反应体系(10mL)：缓冲液磷酸二氢钠-磷酸氢二钠(100mM,pH 7.0)，200mM 1-氰基环己基乙腈，75mg纯酶。反应液于45℃预热10min后，150rpm反应10min。取样500μL上清，加入500μL 2M的HCl终止反应后，利用液相色谱(岛津LC-16)外标法测定转化液1-氰基环己基乙酸转化率。色谱柱为XBridge BEH C 18Column(
Figure PCTCN2019072894-appb-000003
5μm,4.6mm×250mm,1/pkg,Waters)，流动相为缓冲液(0.58g/L磷酸氢二铵，1.83g/L高氯酸钠，高氯酸调节pH为1.8)∶乙腈＝76∶24(v/v)，流速为1mL/min，紫外检测波长215nm，柱温40℃。酶活定义(U)：在45℃、pH 7.0，100mM磷酸二氢钠-磷酸氢二钠缓冲液条件下，每分钟催化生成1μmol1-氰基环己基乙酸所需要的酶量定义为1U。结果见图1所示。
实施例7：腈水解酶突变体45℃下温度稳定性的测定
对实施例5中纯化后的蛋白进行热稳定性的测定。取一定量的蛋白于50mL无菌聚丙烯离心管中，保存于45℃恒温水浴锅中。于不同时间取出蛋白，按照实施例6的方法，测定蛋白的活力。 以未保存时，蛋白的活力为对照，计算各时间下，蛋白的相对残余活力(Residual activity)。
结果见图2所示，经测定，原始腈水解酶LNIT5的半衰期为6h，突变体LNIT5-L201F的半衰期为16h，融合蛋白NIT LNIT5-AcN的半衰期为27h。
实施例8：含有腈水解酶突变体的重组大肠杆菌活力的测定
将实施例4中培养得到的含有腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET-28b(+)-LNIT5，E.coli BL21(DE3)/pET-28b(+)-LNIT5-L201F和E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN进行活力的测定。腈水解酶活力检测反应体系(10mL)：缓冲液磷酸二氢钠-磷酸氢二钠(200mM,pH 7.0)，终浓度100mM 1-氰基环己基乙腈，重组大肠杆菌湿菌体10g/L。反应液于45℃预热10min后，150rpm反应10min。取样500μL上清，利用液相色谱(岛津LC-16)外标法测定转化液1-氰基环己基乙酸转化率。液相检测条件如实施例6中所述，结果见图3所示。
实施例9：含有腈水解酶突变体的重组大肠杆菌45℃下温度稳定性的测定
将实施例4中培养得到的含有腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET-28b(+)-LNIT5，E.coli BL21(DE3)/pET-28b(+)-LNIT5-L201F和E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN静息细胞用缓冲液磷酸二氢钠-磷酸氢二钠(200mM,pH 7.0)配置成20g/L的菌悬液，保存于45℃恒温水浴锅中。于不同时间取出菌悬液，按照实施例8的方法，测定静息细胞的活力。以未保存时，静息细胞的活力为对照，计算各时间下，静息细胞的相对残余活力，结果见表2所示。
表2含有腈水解酶突变体的重组大肠杆菌静息细胞在45℃下的稳定性
Figure PCTCN2019072894-appb-000004
实施例10：使用含腈水解酶突变体的重组大肠杆菌转化400mM 1-氰基环己基乙腈
分别称取0.5g实施例4方法获得的E.coli BL21(DE3)/pET-28b(+)-LNIT5，E.coli BL21(DE3)/pET-28b(+)-LNIT5-L201F和E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN湿菌体悬浮于10mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入0.592g 1-氰基环己基乙腈(终浓度400mM)，45℃恒温水浴反应。于不同时间取样，12000rpm离心，弃去沉淀。处理后的反应液用高效液相色谱分析产物浓度。高效液相色谱分析条件如实施例6中所述。
经测定，如图4所述，E.coli BL21(DE3)/pET-28b(+)-LNIT5，E.coli BL21(DE3)/pET-28b(+)-LNIT5-L201F和E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN均可在6h内将底物反应完全，其中E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN快于E.coli BL21(DE3)/pET-28b(+)-LNIT5。
实施例11：使用含腈水解酶突变体NIT LNIT5-AcN的重组大肠杆菌转化750mM 1-氰基环己基乙腈
分别称取0.5g实施例4方法获得的E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN湿菌体悬浮于10mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入1.11g 1-氰基环己基乙腈(终浓度0.75M)，45℃恒温水浴反应。于不同时间取样，12000rpm离心3min，弃去沉淀。处理后的反应液用高效液相色谱分析产物浓度。高效液相色谱分析条件如实施例6中所述。
经测定，如图5所述，突变体E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN可在8h内将底物反应完全，远快于E.coli BL21(DE3)/pET-28b(+)-LNIT5。
实施例12：使用含腈水解酶突变体NIT LNIT5-AcN的重组大肠杆菌转化1.0M 1-氰基环己基乙腈
分别称取0.5g实施例4方法获得的E.coli BL21(DE3)/pET28b(+)-NIT LNIT5-AcN湿菌体悬浮于10mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入1.48g 1-氰基环己基乙腈(终浓度1.0M)，45℃恒温水浴反应。于不同时间取样，12000rpm离心3min，弃去沉淀。处理后的反应液用高效液相色谱分析产物浓度。高效液相色谱分析条件如实施例6中所述。
经测定，如图5所述，突变体E.coli BL21(DE3)/pET28b(+)-NIT LNIT5-AcN可在11h内将底物反应完全。
实施例13：使用固定化细胞转化750mM 1-氰基环己基乙腈
称取2g实施例4方法获得的E.coli BL21(DE3)/pET-28b(+)-NIT LNIT5-AcN湿菌体悬浮于20mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入终浓度0.006g/mL硅藻土，室温搅拌1h。随后加入终浓度3％(v/v)聚乙烯亚胺(质量浓度5％(w/w)水溶液形式加入)，室温搅拌1h。最后加入终浓度1％(v/v)戊二醛(25％(w/w)水溶液形式加入)，搅拌1h，真空抽滤获得固定化细胞。
将上述得到的全部固定化细胞(固定化细胞用量以静息细胞计为100g/L)悬浮于20mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入2.22g 1-氰基环己基乙腈(终浓度750mM)，45℃恒温水浴反应，每批次反应8小时。每批反应结束后，进行真空抽滤固液分离，反应液用高效液相色谱分析产物浓度(见实施例6)，固定化细胞则投入下一批次反应。结果，所制得的固定化细胞重复利用批次为6批，每批次转化率都大于99％。

Claims (10)

  1. 一种腈水解酶突变体，其特征在于所述突变体是将SEQ ID No.2所示氨基酸序列的第201位进行突变，或对第324-381位氨基酸序列进行替换获得的。
  2. 如权利要求1所述腈水解酶突变体，其特征在于所述突变体为下列之一：(1)将SEQ ID No.2所示氨基酸序列的第201位亮氨酸突变为苯丙氨酸；(2)将SEQ ID No.2所示氨基酸序列第324-381位氨基酸替换为SEQ ID No.8所示来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044腈水解酶的第324-371位氨基酸。
  3. 如权利要求1所述腈水解酶突变体，其特征在于所述突变体氨基酸序列为SEQ ID No.4或SEQ ID No.6所示。
  4. 一种权利要求1所述腈水解酶突变体的编码基因。
  5. 一种由权利要求4所述编码基因构建的重组载体。
  6. 一种由权利要求5所述重组载体转化获得的重组基因工程菌。
  7. 一种权利要求1所述腈水解酶突变体在催化1-氰基环己基乙腈制备1-氰基环己基乙酸中应用。
  8. 如权利要求7所述的应用，其特征在于所述的应用以含腈水解酶突变体编码基因工程菌经发酵培养获得的湿菌体、湿菌体固定化细胞或者湿菌体超声破碎后提取的纯酶为催化剂，以1-氰基环己基乙腈为底物，以pH＝7.0、200M磷酸氢二钠-磷酸二氢钠缓冲液为反应介质构成反应体系，45℃恒温水浴反应完全后，将反应液分离纯化，获得1-氰基环己基乙酸。
  9. 如权利要求8所述的应用，其特征在于所述反应体系中，底物终浓度以缓冲液体积计为100～1300mM，所述催化剂用量以湿菌体重量计为10～100g/L缓冲液。
  10. 如权利要求8所述的应用，其特征在于所述催化剂按如下方法之一制备：(1)将含腈水解酶突变体编码基因工程菌接种到LB培养基中，37℃培养10-12小时，按体积浓度2％的接种量接种至含终浓度50mg/L卡那霉素的LB培养基进行扩大培养，37℃培养至培养液的OD 600为0.6-0.8之间，加入终浓度为0.1mM异丙基-β-D-硫代吡喃半乳糖苷，28℃诱导培养10小时，离心，收集菌体，用生理盐水清洗2次，得到湿菌体；(2)将步骤(1)湿菌体用含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液重悬，超声波破，破碎产物离心后取上清液作为粗酶液；将粗酶液以1mL/min的流速通过经平衡缓冲液冲洗的Ni-NTA柱，用洗脱缓冲液洗脱弱吸附的杂蛋白，流速为2mL/min；再用蛋白洗脱缓冲液洗脱并收集目的蛋白，流速为2mL/min；最后将收集的目的蛋白以质量浓度0.9％氯化钠水溶液为透析液进行透析，取截留液即为纯酶；所述平衡缓冲液为含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液；所述洗脱缓冲液为含终浓度300mM NaCl和50mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液；所述蛋白洗脱缓冲液为含终浓度300mM NaCl和250mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液；(3)将步骤(1)湿菌体悬浮于pH＝7.0、200mM磷酸氢二钠-磷酸二氢钠缓冲液体系中，加入终浓度6mg/mL硅藻土，室温搅拌1h，随后加入体积终浓度3％聚乙烯亚胺，室温搅拌1h，最后加入体积终浓度1％戊二醛，搅拌1h，真空抽滤，获得固定化细胞；所述聚乙烯亚胺以质量浓度5％水溶液形式加入，所述戊二醛以质量浓度25％水溶液形式加入。
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