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Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate

A technology of nitrilase and mutants, applied in the directions of immobilization on or in inorganic carriers, hydrolase, immobilization on/in organic carriers, etc., can solve the problems of poor thermal stability and low catalytic activity of catalyst enzymes

Active Publication Date: 2020-05-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high solubility of the substrate 1-cyanocyclohexyl acetonitrile under the condition of high temperature, the catalytic reaction can be promoted, but the thermal stability of the catalyst enzyme is poor, and the catalytic activity is low under high temperature conditions, so the existing Nitrilase cannot meet the requirements, and it is necessary to improve the thermal stability of nitrilase through molecular modification, thereby improving catalytic efficiency and realizing industrial production

Method used

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  • Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
  • Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate
  • Nitrilase mutant and application thereof in preparation of anti-epileptic drug intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Semi-rational design and site-directed mutation

[0039] To contain the nitrilase gene AcN-M cloned in Acidovorax facilis (A.facilis) CCTCC NO: M 209044 (shown in the nucleotide sequence of SEQ ID NO.1, the encoded protein amino acid sequence is shown in SEQ ID NO.2 )'s pET-28b(+)-AcN-M plasmid as a template, calculate the sites that can improve thermal stability through http: / / kazlab.umn.edu / , and then perform site-directed mutation of the whole plasmid (Table 1) PCR amplification . PCR reaction system (50 μL): template 0.5-20ng, 2×Phanta max Buffer 25 μL, 0.2 mM dNTP, primers 0.2 μM, Phanta Max Super-Fidelity DNA Polymerase 1 μL, add water to make up to 50 μL. PCR conditions: (1) Pre-denaturation at 95°C for 3 minutes; (2) Denaturation at 95°C for 15 seconds; (3) Annealing at 60°C for 15 seconds; (4) Extension at 72°C for 5.5 minutes, steps (2) to (4) for a total of 30 cycles; (5) Finally, extend at 72°C for 10 minutes and store at 16°C. The PCR product ...

Embodiment 2

[0044] Example 2: Expression of nitrilase mutants

[0045] The mutant E.coli BL21(DE3) / pET28b(+)-AcN-T151V obtained in Example 1, E.coli BL21(DE3) / pET28b(+)-AcN-C223A, E.coli BL21(DE3) / pET28b(+)-AcN-C250G, and the combined mutant E.coli BL21(DE3) / pET28b(+)-AcN-T151V / C223A / C250G, and the original strain E.coli BL21(DE3) / pET28b(+)- AcN-M was inoculated into LB medium, cultured at 37°C for 10-12 hours, inoculated into LB medium containing kanamycin (final concentration 50 mg / L) at 2% inoculum volume, and expanded to culture medium at 37°C OD 600 between 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and induce culture at 28°C for 10 hours. The bacterial cells were collected by centrifugation of the culture solution, washed twice with normal saline, and the corresponding wet bacterial cells were obtained.

Embodiment 3

[0046] Embodiment 3: the purification of nitrilase mutant protein

[0047] (1) Add equilibrium buffer (50mM NaH 2 PO 4 , 300mM NaCl buffer, pH 8.0) after resuspending the cells, ultrasonic disruption (400W, 25min, 1s disruption 1s pause). The crushed product was centrifuged (12000×g, 10 min), and the supernatant was taken as a crude enzyme solution for separation and purification.

[0048] (2) After prepacking 20mL Ni-NTA affinity chromatography column, use equilibration buffer (50mM NaH 2 PO 4 , 300mMNaCl, pH 8.0) for equilibration at a flow rate of 2mL / min.

[0049] (3) After cleaning 8-10 column volumes, pass the obtained crude enzyme solution through the Ni-NTA column at a flow rate of 1 mL / min, and the target protein is mounted on the chromatography column. After loading, a large number of unadsorbed foreign proteins will not be bound to the resin and will be removed directly.

[0050] (4) Use elution buffer (50mM NaH 2 PO 4 , 300mM NaCl, 50mM imidazole, pH 8.0) t...

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Abstract

The invention discloses a nitrilase mutant and an application thereof in preparation of an anti-epileptic drug intermediate. The mutant is obtained by mutating one or more of 151th amino acid, 223th amino acid and 250th amino acid of an amino acid sequence shown in SEQ ID No.2. The thermal stability of the nitrilase mutant AcN-T151V / C223A / C250G is improved by 1.73 times, 1M 1-cyanocyclohexyl acetonitrile is hydrolyzed by use of recombinant escherichia coli containing the nitrilase mutant at the temperature of 35 DEG C to generate 1-cyanocyclohexylacetic acid, and the yield of a final product reaches 95%. When 1.2 M 1-cyanocyclohexyl acetonitrile is hydrolyzed at the temperature of 35 DEG C, the final yield reaches 97%. Gabapentin is synthesized by use of the nitrilase mutant, and the yieldof the final product reaches 80%.

Description

[0001] (1) Technical field [0002] The invention relates to a mutant derived from nitrilase of Acidovorax facilis CCTCC NO:M 209044 and its application in the synthesis of 1-cyanocyclohexylacetic acid, an intermediate of antiepileptic drugs. [0003] (2) Background technology [0004] Gabapentin is a new type of antiepileptic drug first developed by Warner-Lambert Company of the United States. Compared with similar drugs currently on the market, it has the advantages of fast oral absorption, less toxic and side effects, obvious therapeutic effect and strong tolerance. The blood-brain barrier of the human brain is less likely to interact with other antiepileptic drugs, and its effect as a superimposed drug for intractable epilepsy is particularly prominent. [0005] 1-cyanocyclohexylacetic acid is a key intermediate in the synthesis of antiepileptic drug gabapentin, and it has broad application prospects in the market. At present, the industrial synthesis of gabapentin and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12N1/21C12N15/70C12N11/14C12N11/082C12P13/00C12R1/19
CPCC12N9/78C12N15/70C12N11/14C12N11/08C12P13/002C12Y305/05001C12R2001/01C12R2001/19
Inventor 薛亚平熊能吕佩锦郑裕国
Owner ZHEJIANG UNIV OF TECH
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