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Nitrilase mutant and its application

A technology of nitrilase and mutants, applied in nitrilase mutants and its application fields, can solve the problems of low reaction efficiency, long reaction time, poor temperature stability, etc., achieve high application value, and improve the activity of cyanocarboxylic acid Effect

Active Publication Date: 2020-07-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the existing biocatalysts still have poor temperature stability, low catalytic reaction efficiency at high temperatures, and long reaction times, the solubility of the substrate 1-cyanocyclohexyl acetonitrile is significantly affected by temperature. The reaction at higher temperature can increase the solubility of the substrate and promote the reaction efficiency, but the existing nitrilases cannot meet this requirement

Method used

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  • Nitrilase mutant and its application
  • Nitrilase mutant and its application
  • Nitrilase mutant and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Construction of a nitrilase mutant library

[0032] The pET-28b ( +)-AcN-F168V plasmid was used as a template, and T7promoter and T7terminator were used as primers (Table 1) for PCR amplification, and mutations were randomly introduced. PCR reaction system (50μL): template 0.5~20ng, 1×Taq Buffer (without Mg 2+ ), 0.2mM dNTP, 0.3mMnCl 2 , 2mM MgCl 2 , primers T7promoter and T7terminator each 0.2μM, Taq DNA polymerase 5U. PCR conditions (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 94°C for 50 s; (3) annealing at 55°C for 60 s; (4) extension at 72°C for 120 s, a total of 30 cycles of steps (2) to (4); (5) ) and finally extended at 72°C for 5min, and stored at 4°C. The PCR products were analyzed by agarose gel electrophoresis and recovered by cutting the gel.

[0033] Subsequently, the product recovered from the above-mentioned gel was used as a primer to amplify to obtain a complete plasmid. PCR system (50μL) is 5×PrimeSTAR Buffer (Mg 2+ p...

Embodiment 2

[0038] Example 2: High throughput screening of mutants

[0039] Pick a single colony in Example 1 and culture it in a 96-deep well plate, add 1000 μL LB liquid medium (containing a final concentration of 50 μg / mL kanamycin, and a final concentration of 1 mM IPTG) to each well plate, and incubate at 37° C. for 18 hours. Centrifuge the cells in the 96 deep-well plate for 30 min (3000 rpm, 4°C), discard the supernatant, and resuspend the cells with 1.5 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (200 mM, pH 7.0). Take 500 μL of bacterial suspension into a new 96-deep well plate, add substrate 1-cyanocyclohexylacetonitrile (final concentration 100 mM) to each well, and react at 35° C. for 1 h. Take 500 μL of the bacterial suspension into a new 96-deep well plate, add the substrate 1-cyanocyclohexylacetic acid (final concentration 100 mM) to each well, and react at 35° C. for 12 h. The rest of the bacterial solution was placed in a constant-temperature wat...

Embodiment 3

[0042] Example 3: Site-directed mutagenesis and screening

[0043] Using the expression plasmid pET-28b(+)-AcN-F168V as a template, site-directed mutagenesis was performed by amplification of the whole plasmid. The PCR system (50 μL) is: template 0.5~20ng, primers T201-f and T201-r (see Table 1 for sequence) each 10-15pmol, 5×PrimeSTAR Buffer (Mg 2+ plus), 0.2mM dNTPs, 1.25 U PrimeSTAR HS DNA Polymerase. PCR conditions (1) Pre-denaturation at 98°C for 3min; (2) Denaturation at 98°C for 10s; (3) Annealing at 55°C for 5s; (4) Extension at 72°C for 6.3min, steps (2) to (4) totaled 30 cycles; ( 5) Finally, extend at 72°C for 5 minutes and store at 4°C. The PCR product was verified by agarose gel electrophoresis, digested with DpnI, introduced into E.coli BL21(DE3), and applied to an LB plate containing 50 μg / mL kanamycin to obtain a single clone. In this round of site-directed saturation mutation, a total of 200-300 single clones were generated, which were sequenced and contain...

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Abstract

The invention discloses nitrilase mutant and application thereof. The mutant is obtained by carrying out mutation one or more of the sits including 201 site, 339 site and 343 site of an amino acid sequence as shown in SEQ ID No.2. By modification of protein molecules, the thermal stability of nitrilase AcN pure enzyme is improved by 14 times to a maximum extent, recombinant escherichia coli which contains the nitrilase mutant is used for hydrolyzing 1-cyanocyclohexane acetonitrile at the high temperature (45 DEG C), and the reaction time is shortened to be one third of the original reaction time. Therefore, the acquired mutant has good application prospect in efficient catalysis of the 1-cyanocyclohexane acetonitrile to synthesize 1-cyanocyclohexane acetic acid as a gabapentin intermediate.

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 an anti-epileptic drug first developed by Warner-Lambert Company of the United States. Compared with similar products currently used, it has fast oral absorption, good tolerance, small toxic and side effects, and good therapeutic effect. It does not metabolize in the body and does not interact with plasma proteins. Combined, it does not induce liver enzymes, can pass through the blood-brain barrier of the human brain, and has a low possibility of interacting with other anti-epileptic drugs, especially as a superimposed drug for refractory epilepsy. [0005] 1-Cyanocyclohexaneacetic acid (1-Cyanocyclohexaneacetic acid) is a key intermediate in the synthesis of a new generation of ant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12P13/00
CPCC12N9/78C12P13/002C12Y305/05001
Inventor 薛亚平郑裕国徐喆徐伟亮
Owner ZHEJIANG UNIV OF TECH
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