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Amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid

A mutant, amidase technology, applied in the field of enzyme engineering, can solve the problems of low yield, heavy production process pollution, lengthy synthesis steps and the like, and achieve the effect of improving the activity of the parent

Active Publication Date: 2020-05-15
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, 2-chloronicotinic acid is mainly produced by the 3-cyanopyridine method in industry, but its production process has defects such as heavy pollution, lengthy synthesis steps, and low yield.

Method used

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  • Amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid
  • Amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid
  • Amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Induction and expression purification of amidase

[0030] Step 1: Cultivation of Recombinant Bacteria

[0031] Inoculate 10 μL of the preserved bacterial solution in glycerol tubes into 10 mL of liquid LB medium (containing Kan 50 μg / mL), incubate at 37°C and 200 r / min for 12 hours, then transfer to 100 mL of fresh LB medium containing Kan at 2% inoculum size to continue Grow to OD 600 When it reaches 0.6, add IPTG with a final concentration of 0.1 mM, and induce culture at 28° C. overnight (12 h). After the cultivation, the cells were collected by centrifugation and washed twice with 0.85% saline.

[0032] Step 2: Purification of amidase

[0033] (1) Take 5 g of wet bacteria and suspend them in 50 mL of Tris-HCl buffer (20 mM, pH 8.0), oscillate and shake well, and use an ultrasonic cell disruptor to break the cells (crushing power 40 W, each ultrasonic work 5 s, interval 5 s, total 99 loops);

[0034] (2) After the crushing is completed, take the crush...

Embodiment 2

[0042] Example 2. Site-directed saturation mutation and screening of amidase

[0043] Description of site-directed saturation mutation technology reference (Applied Microbiology and Biotechnology, 2014,98(6):2473-2483), reference of high-throughput screening model for positive mutants (CN100370034; Applied Microbiology and Biotechnology, 2007,74:256-262) description of. The specific process is as follows:

[0044] Step 1: Site-directed mutagenesis

[0045] Carry out saturation mutation at the 378th, 402 and 403 amino acids in the amino acid sequence of the parent pantoea amidase mutant G175A, and design primers A378, V402 and L403 (see Table 1) to clone the pantoea amidase mutation The plasmid pET28-G175A encoding gene of body G175A was used as a template for full plasmid amplification.

[0046] Table 1 Design table of primers for site-directed saturation mutagenesis

[0047]

[0048] Note: N=A / G / C / T, K=G / T, M=A / C.

[0049] The PCR system is: 2×phanta Max buffer 25 μL,...

Embodiment 3

[0055] Example 3. Re-screening of amidase positive mutants

[0056] The positive bacteria obtained in Example 2 were cultured according to the culture conditions of Example 1 to obtain mutant whole cells. Weigh the wet cells and add buffer to mix well to make a bacterial suspension with a wet cell concentration of 40g / L. The reaction system for the determination of mutant viability was: total system 10mL, cell final concentration 0.25g / L, 50mM Tris-HCl (pH 8.0), 50mM 2-chloronicotinamide. Shake at 55°C, 150r / min, react for 10min, take 1mL and add 100μL 2M hydrochloric acid to terminate the reaction, dilute 10 times and use liquid chromatography for detection. Liquid chromatography detection conditions: mobile phase: acetonitrile: water: phosphoric acid = 250:750:1, flow rate 1mL / min, 2-chloronicotinic acid detection wavelength 270nm.

[0057] Enzyme activity unit (U) definition: Under the conditions of 55°C and pH 8.0, the cells required to catalyze 2-chloronicotinamide to p...

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Abstract

The invention discloses an amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid and belongs to the technical field of enzyme engineering. The amidase mutant is obtained through single site mutation or multi-site mutation of a 378th site, a 402nd site or a 403rd site of the amino acid sequence shown in SEQ ID NO.1. The activity of the amidase mutant disclosed by the invention is greatly improved when being compared with that of a parent, and when catalysis is implemented by using a crude extract or engineering bacterium whole cells of the enzyme, the reactionenzyme activity is still kept at a high state. In addition, the amidase mutant disclosed by the invention is applicable to a catalysis temperature of 30-55 DEG C, and a basis is made for production of2-chloronicotinic acid by using an industrial enzyme method.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an amidase mutant and its application in catalytically synthesizing 2-chloronicotinic acid. Background technique [0002] 2-Chloronicotinic acid is an important intermediate compound for the synthesis of pesticides and medicines. In the field of pesticides, 2-chloronicotinic acid can be used to synthesize a series of fungicides of sulfonylurea herbicide nicosulfuron, amide herbicide diflufen, amide fungicide boscalid and triazolethiones Active compounds, etc.; in the field of medicine, 2-chloronicotinic acid is a synthetic anti-AIDS drug - nevirapine, antidepressant drug - mirtazapine, non-steroidal anti-inflammatory analgesics - niflufenamic acid, pranoprofen and nicotine Important intermediates such as acids. Due to the wide application of 2-chloronicotinic acid in pesticides and medicines, its market demand is increasing day by day. [0003] At present, the produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/80C12N15/70C12P17/12C12Y305/01004
Inventor 郑仁朝刘长丰吴哲明郑裕国
Owner ZHEJIANG UNIV OF TECH
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