Nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid

Patent metadata

Field	Value
Publication number	US-12428656-B2
Application number	US-202017603864-A
Country	US
Kind code	B2
Filing date	Dec 11, 2020
Priority date	Feb 28, 2020
Publication date	Sep 30, 2025
Grant date	Sep 30, 2025

Abstract

The present invention provides a nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid, the nitrilase mutant is obtained by mutating one or two of the amino acids at position 180 and 205 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by semi-rational design and protein molecular modification, the specific enzyme activity of the nitrilase double mutant AcN-G180D/A205C was increased by up to 1.6 folds, and the conversion rate>99%. And the reaction time was shortened to a quarter of the original using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at high temperature (50° C.). Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid.

First claim

The invention claimed is:

1. A nitrilase mutant, wherein the mutant has nitrilase activity of catalyzing the conversion of the substrate dinitrile to a product of monocyano carboxylic acid compound, and the nitrilase mutant comprises the amino acid sequence of SEQ ID NO: 2, except for the substitution G180D, the substitution G180F, the substitution A205C, or the substitutions G180D and A205C.

2. A polynucleotide comprising a nucleotide sequence encoding the nitrilase mutant of claim 1 .

3. A recombinant genetically engineered E. coli host cell transformed with the polynucleotide of claim 2 .

4. A method for producing 1-cyanocyclohexyl acetic acid, the method comprising:
reacting a catalyst and a substrate in a reaction medium to produce a reaction solution comprising 1-cyanocyclohexyl acetic acid,
wherein the catalyst is wet cells comprising the nitrilase mutant of claim 2 ,
wherein the wet cells are obtained by fermentation culture of a genetically engineered E. coli cell expressing the nitrilase mutant, immobilized E. coli cells comprising the nitrilase mutant or the nitrilase mutant,
wherein the nitrilase mutant is purified,
wherein the purified nitrilase is obtained by subjecting the wet cells to ultrasonic breaking and centrifugation,
wherein the substrate is 1-cyanocyclohexylacetonitrile,
wherein the reaction medium is a pH 7.0, 200 mM disodium hydrogen phosphate-sodium dihyrdrogen phosphate buffer,
wherein the reaction is carried out in a constant temperature water bath at 25-50° C., and
wherein, after the reaction is completed, the reaction solution is subjected to separation and purification to obtain the 1-cyanocyclohexyl acetic acid.

5. The method of claim 4 , wherein the final concentration of the substrate in the reaction medium is 5-1000 mM, and
wherein catalyst is wet cells and the concentration of the wet cells in the reaction medium is 10-100 g/L.

6. The method of claim 4 , wherein the wet cells are prepared according to the following method:
culturing an LB medium with the genetically engineered E. coli host expressing the nitrilase mutant at 37° C. for 10-12 hours to produce an inoculum;
inoculating the LB medium with the inoculum with a 1% incubating volume, wherein the LM medium contains 50 mg/L kanamycin and culturing at 37° C.;
inducing an expression of the nitrilase mutant by adding isopropyl-p-D-thiogalactopyranoside to a final concentration of 0.1 mM when the OD 600 of the culture reaches 0.6-0.8 and culturing at 28° C. for 10 hours;
harvesting cells by centrifugation; and
washing the cells with normal saline twice, thereby obtaining the wet cells.

7. The method of claim 4 , wherein the purified nitrilase is prepared according to the following method:
resuspending the wet cells with a pH 7.0, 100 mM NaH 2 PO 4 —Na 2 HPO 4 buffer containing 300 mM NaCl;
ultrasonically breaking the resuspended wet cells under the conditions of 400 W, 25 min, 1 s breaking and 1 s pause;
centrifuging the broken cells at 8000 rpm for 15 min to obtain a supernatant, wherein the supernatant is a crude enzyme solution;
applying the crude enzyme solution to a Ni-NTA column that has been washed with an equilibrium buffer, wherein the equilibrium buffer is a pH 8.0, 50 mM NaH 2 PO 4 buffer containing 300 mM NaCl, and 50 mM imidazole, pH 8.0;
applying a first elution buffer to the Ni-NTA column with the applied crude enzyme solution at a flow rate of 2 mL/min to elute weakly absorbed protein impurities, wherein the first elution buffer is pH 8.0, 50 mM NaH 2 PO 4 buffer containing 300 mM NaCl, and 50 mM imidazole;
applying a second elution buffer to the Ni-NTA column with the first elution buffer at a flow rate of 2 mL/min to elute and collect the nitrilase mutant, wherein the second elution buffer is pH 8.0, 50 mM NaH 2 PO 4 buffer containing 300 mM NaCl, and 50 mM imidazole; and
dialyzing the collected nitrilase mutant with with a 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Xiong Neng
• Li Qian
• Zheng Yuguo

Classifications

• C12Y305/05001

  Nitrilase (3.5.5.1) · CPC title

• C12N2800/101

  for bacteria · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/78 Primary

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12R2001/19

  Escherichia coli · CPC title