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Pantoea amidase mutant, gene, engineering bacterium and application thereof

A technology of bacteramidase and mutants, applied in genetic engineering, application, plant gene improvement, etc., can solve the problems of low catalytic efficiency, achieve high catalytic efficiency, facilitate extraction and purification, and reduce the effect of product inhibition

Active Publication Date: 2018-04-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing research reports have shown that the catalytic efficiency of amidase hydrolysis of 2-chloronicotinamide is generally low

Method used

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  • Pantoea amidase mutant, gene, engineering bacterium and application thereof
  • Pantoea amidase mutant, gene, engineering bacterium and application thereof
  • Pantoea amidase mutant, gene, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Site-directed saturation mutagenesis and screening of amidases

[0023]Description of site-directed saturation mutagenesis technology reference (Appl.Microbiol.Biotechnol., 2014, 98, 2473-2483), high-throughput screening model reference for positive mutants (CN100370034; Appl. Microbiol. Biotechnol., 2007, 74, 256-262) description of. The specific process is as follows:

[0024] To the pantoea amidase Pa-Ami (the amino acid sequence is shown in SEQ ID No.2, the nucleotide sequence is shown in SEQ ID No.1) in the amino acid sequence derived from Pantoea sp. (GenBank No.WP008109374). Glycine at position 175 (Gly, G), threonine at position 301 (Thr, T), alanine at position 305 (Ala, A) and serine at position 309 (Ser, S) were For saturation mutation, each mutation primer (see Table 1) was designed, and the plasmid pET28-Pa-Ami cloned with the gene encoding panbiotic amidase Pa-Ami was used as a template, and the whole plasmid was amplified. PCR system: 25μL o...

Embodiment 2

[0028] Example 2: Multisite stacking saturation mutagenesis of amidase mutant G175A

[0029] The saturation mutation technology and the high-throughput screening method for positive mutants are the same as those in Example 1. The specific process is as follows:

[0030] On the basis of the amidase mutant G175A, the sites including the 301 threonine protrusion (Thr, T), the 305th alanine (Ala, A) and the 309th serine (Ser, S) were site-directed For saturation mutation, whole plasmid amplification was performed using the plasmid pET28-G175A of the amidase mutant G175A as a template. PCR system: 2×phanta Max buffer 25 μL, dNTPmixture (10 mM) 0.75 μL, mutation upstream primer (50 μM) and downstream primer (50 μM) (see Table 1 for primer sequences) each 1 μL, plasmid pET28-G175A 0.5 μL, Phanta Max DNA Polymerase 0.5 μL, ddH 2 O supplemented to 50 μL. PCR conditions were pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 15 s, annealing at 55-65 °C for 15 s, extensio...

Embodiment 3

[0031] Example 3: Induced expression and purification of parent amidase and amidase mutant engineered bacteria

[0032] (1) Induced expression

[0033] The starting strain E.coli BL21(DE3) / pET28-Pa-Ami and mutant strains E.coli BL21(DE3) / pET28-G175A (Example 1), E.coli BL21(DE3) / pET28-G175A / A305T will be included (Example 2) and each positive mutant strain were inoculated into LB liquid medium containing 50 mg / L kanamycin, and cultured at 37°C and 150 r / min for 12 h, followed by 1% (v / v) The inoculum was transferred to fresh LB liquid medium containing 50 mg / L kanamycin, and cultured at 37 °C and 150 r / min to the bacterial concentration OD 600 Then add IPTG with a final concentration of 0.1 mM to the medium, induce and culture at 28 °C, 150 r / min for 12 h, take the culture and centrifuge the culture at 4 °C and 8000 rpm for 15 min to collect wet cells, which can be used for enzyme activity determination and Biocatalytic preparation of 2-chloronicotinic acid.

[0034] (2) Pr...

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Abstract

The invention discloses a pantoea amidase mutant, a gene, an engineering bacterium and an application thereof. The amidase mutant is obtained by conducting single mutation or multiple mutation on the105th, 175th, 301st, 305th or 309th site of an amino acid sequence of pantoea amidase shown as SEQ ID No.2. The activity of the pantoea amidase mutant provided by the invention, in comparison with a parent, is improved by 2-3 times; a tolerance capacity to a substrate, namely 2-chloronicotinamide, exceeds 200mM, and the tolerance capacity can even exceed 1M by virtue of a material supplementing method; and a conversion rate can be still kept above 95%. With the application of the material supplementing method provided by the invention, an inhibitory effect of 2-chloronicotinic acid to an amidase product or bacterium product can be effectively relieved, catalysis efficiency can be kept at relatively high level, and meanwhile, the separation-out of the finished product, namely the 2-chloronicotinic acid, can be promoted to the greatest extent, so that the extraction and purification of the finished product are facilitated. Therefore, the amidase mutant provided by the invention is applicable to the enzymatic industrial production of the 2-chloronicotinic acid.

Description

(1) Technical field [0001] The present invention relates to a preparation method of 2-chloronicotinic acid, in particular to a panbiotic amidase mutant, an encoding gene, and a method for preparing pesticide and pharmaceutical intermediate 2-chloronicotinic acid by hydrolyzing 2-chloronicotinamide Applications. (2) Background technology [0002] 2-Chloronicotinic Acid (2-CA for short), also known as 2-chloro-3-picolinic acid, is a 2-chlorinated compound of nicotinic acid. 2-Chloronicotinic acid is an important fine chemical intermediate widely used in pesticide and pharmaceutical industries. In the field of pesticides, 2-chloronicotinic acid can be used to synthesize fungicides, insecticides and herbicides, such as nicosulfuron, diflufenacil, etc. (Pesticide, 2003, 42, 5-8; Pesticide, 2013, 565 -567); in the field of medicine, it can be used to synthesize a variety of antibiotics, cardiovascular disease treatment drugs, etc., such as the anti-AIDS drug nevirapine, the anti...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/80C12P17/12C12Y305/01004
Inventor 郑仁朝郑裕国金建强吴哲明汤晓玲
Owner ZHEJIANG UNIV OF TECH
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