Nitrilase mutants and application thereof

Patent metadata

Field	Value
Publication number	US-11001823-B2
Application number	US-201916619344-A
Country	US
Kind code	B2
Filing date	Jan 24, 2019
Priority date	Feb 14, 2018
Publication date	May 11, 2021
Grant date	May 11, 2021

Abstract

The present invention discloses a nitrilase mutant and application thereof. The mutant is obtained by mutating the amino acid at position 201 or replacing one or more amino acids at region 324-381 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by the protein molecular modification, thermostability of the purified nitrilase LNIT5 is increased by up to 4.5 folds; and by utilizing recombinant E. coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at a high temperature (45° C.), product tolerance is increased, activity of NIT5-L201F is increased by 20%, and the mutant NITLNIT5-AcN can completely hydrolyze 750 mM 1-cyanocyclohexylacetonitrile within 8 hours and achieve an doubled conversion rate. 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. In the present invention, by protein molecular modification, thermal stability of pure nitrilase LNIT5 at 45° C. is increased up to 4.5 times, and while 1-cyanocyclohexylacetonitrile is hydrolyzed using recombinant Escherichia coli containing nitrilase mutant at high temperature (45° C.), the product yield is increased. Therefore, the mutants obtained in the present invention have a good application prospect in highly efficiently catalyzing 1-cyanocyclohexylacetonitrile to 1-cyanocyclohexyl acetic acid, the gabapentin intermediate.

First claim

The invention claimed is:

1. A nitrilase mutant having the amino acid sequence of SEQ ID No:6.

2. A method for catalyzing 1-cyanocyclohexylacetonitrile to 1-cyanocyclohexyl acetic acid, comprising providing the nitrilase mutant of claim 1 as a catalyst.

3. The method of claim 2 , further comprising:
providing wet cells, wet cell-immobilized cells or purified nitrilase mutant as the catalyst, 1-cyanocyclohexylacetonitrile as a substrate, and pH=7.0, 200 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer as a reaction medium,
carrying out the reaction in a 45° C. constant temperature water bath,
after the reaction is completed, subjecting the reaction solution to separation and purification to obtain 1-cyanocyclohexyl acetic acid,
wherein the wet cells are obtained by fermenting culture of genetically engineered cells containing a gene encoding the nitrilase mutant, and the purified nitrilase mutant is obtained by subjecting the wet cells to ultrasonic breaking and then extraction.

4. The method of claim 3 , wherein in the reaction system, a final concentration of the substrate calculated by a volume of the buffer is 100˜1300 mM, and an amount of the catalyst calculated by a weight of the wet cells is 10˜100 g/L buffer.

5. The method of claim 3 , wherein the catalyst is prepared according to one of the following methods:
(1) genetically engineered cell containing the gene encoding the nitrilase mutant is inoculated into a LB medium and cultured at 37° C. for 10-12 hours, then the resulting inoculum is inoculated with 2% incubating volume to a LB medium containing kanamycin with a final concentration of 50 mg/L, amplified and cultured at 37° C.; when OD600 of the culture medium reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside is added with a final concentration of 0.1 mM, and the bacteria solution is subjected to induced expression at 28° C. for 10 hours; the wet cells are harvested by centrifugation and washed with normal saline twice;
(2) the wet cells obtained in step (1) are resuspended with 50 mM NaH 2 PO 4 buffer (pH 8.0) containing NaCl with a final concentration of 300 mM, ultrasonic broken, and followed by centrifugation to remove cell debris, the resulting supernatant is a crude enzyme solution; the crude enzyme solution is applied onto the Ni-NTA column which has been washed with equilibrium buffer at a flow rate of 1 mL/min, then the weakly adsorbed protein impurities are eluted with elution buffer at a flow rate of 2 mL/min; then the target protein is eluted with protein elution buffer at a flow rate of 2 mL/min and collected; finally, the obtained target protein is dialyzed with a sodium chloride aqueous solution with a mass concentration of 0.9% as the dialysate, and the retention is purified nitrilase mutant; in which the equilibrium buffer is 50 mM NaH 2 PO 4 buffer (pH 8.0) containing NaCl with a final concentration of 300 mM, the elution buffer is 50 mM NaH 2 PO 4 buffer (pH 8.0) containing NaCl and imidazole with a final concentrations of 300 mM and 50 mM, and the protein elution buffer is 50 mM NaH 2 PO 4 buffer (pH 8.0) containing NaCl and imidazole with a final concentrations of 300 mM and 250 mM; or
(3) the wet cells of the step (1) are suspended in 200 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.0), diatomite is added into the suspension with a final concentration of 0.006 g/mL, and the mixture is stirred at room temperature for 1 h; subsequently, polyethyleneimine is added into the mixture with a final volume concentration of 3%, and the mixture is stirred at room temperature for 1 hour; finally, glutaraldehyde is added with a final volume concentration of 1% and the mixture is stirred for 1 hour, and the immobilized cells are obtained by vacuum filtration; in which, the polyethyleneimine is added in a form of a aqueous solution with a mass concentration of 5%, and the glutaraldehyde is added in a form of a aqueous solution with a mass concentration of 25%.

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Zheng Yuguo
• Xu Zhe
• Liu Zhiqiang

Classifications

• C12P13/002

  Nitriles (-CN) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12Y305/05001

  Nitrilase (3.5.5.1) · CPC title

• C12N9/78 Primary

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