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A kind of pantophyte amidase mutant, gene, engineering bacteria and application thereof

A bacterial amidase and mutant technology, applied in genetic engineering, application, plant genetic improvement and other directions, can solve problems such as low catalytic efficiency, achieve high catalytic efficiency, reduce product inhibition effect, and facilitate extraction and purification.

Active Publication Date: 2020-10-02
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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  • A kind of pantophyte amidase mutant, gene, engineering bacteria and application thereof
  • A kind of pantophyte amidase mutant, gene, engineering bacteria and application thereof
  • A kind of pantophyte amidase mutant, gene, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Site-directed saturation mutation and screening of amidase

[0028] Description of site-directed saturation mutation technology reference (Appl.Microbiol.Biotechnol., 2014,98,2473-2483), reference for high-throughput screening model of positive mutants (CN100370034; Appl.Microbiol.Biotechnol., 2007,74,256-262) description of. The specific process is as follows:

[0029] In the amino acid sequence of Pantoea amidase Pa-Ami (the amino acid sequence is shown in SEQ ID No.2, and the nucleotide sequence is shown in SEQ ID No.1) derived from Pantoea sp. (GenBank No.WP008109374) Glycine (Gly, G) at position 175, threonine at position 301 (Thr, T), alanine at position 305 (Ala, A) and serine at position 309 (Ser, S) were respectively For saturation mutation, design primers for each mutation (see Table 1), and use the plasmid pET28-Pa-Ami cloned with the gene encoding Pantoea amidase Pa-Ami as a template to amplify the whole plasmid. PCR system: 2×phanta Max buffer...

Embodiment 2

[0033] Example 2: Multi-site stacking saturation mutation of amidase mutant G175A

[0034] The saturation mutation technique and the high-throughput screening method for positive mutants are the same as in Example 1. The specific process is as follows:

[0035] On the basis of the amidase mutant G175A, sites including the 301th threonine (Thr, T), the 305th alanine (Ala, A) and the 309th serine (Ser, S) were determined For saturation mutation, the plasmid pET28-G175A of the amidase mutant G175A was used as a template for full plasmid amplification. The PCR system is: 2×phanta Max buffer 25 μL, dNTP mixture (10 mM) 0.75 μL, mutant upstream primer (50 μM) and downstream primer (50 μM) (see Table 1 for primer sequence) each 1 μL, plasmid pET28-G175A 0.5 μL, Phanta Max DNA Polymerase 0.5 μL, ddH 2 O to make up 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, extension at 72°C for 6.5 min, and 30 cyc...

Embodiment 3

[0036] Example 3: Induced expression and purification of parent amidase and amidase mutant engineered bacteria

[0037] (1) Induced expression

[0038] Will include starting strain E.coli BL21(DE3) / pET28-Pa-Ami and mutant strain E.coli BL21(DE3) / pET28-G175A (Example 1), E.coli BL21(DE3) / pET28-G175A / A305T (Example 2) each positive mutant bacterial strain is inoculated respectively in the LB liquid culture medium containing 50mg / L kanamycin, cultivates 12h under 37 ℃, 150r / min, then with 1% (v / v) Transfer the inoculum to fresh LB liquid medium containing 50mg / L kanamycin, and cultivate at 37°C and 150r / min until the cell concentration OD 600 0.4 to 0.8, then add IPTG with a final concentration of 0.1mM to the medium, induce culture at 28°C and 150r / min for 12h, take the culture and centrifuge at 8000rpm for 15min at 4°C, and collect the wet cells, which can be used for enzyme activity determination and Biocatalytic preparation of 2-chloronicotinic acid.

[0039] (2) Protein p...

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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

[0001] (1) Technical field [0002] The invention relates to a preparation method of 2-chloronicotinic acid, in particular to a pantotheca amidase mutant, a coding gene, and its use in hydrolyzing 2-chloronicotinamide to prepare pesticides and pharmaceutical intermediates 2-chloronicotinic acid Applications. [0003] (2) Background technology [0004] 2-Chloronicotinic acid (2-Chloronicotinic Acid, 2-CA for short), also known as 2-chloro-3-pyridinecarboxylic acid, belongs to the 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-methyl, difluzachlor, etc. (Pesticides, 2003, 42, 5-8; Pesticides, 2013, 565 -567); in the field of medicine, it can be used to synthesize a variety of antibiotics and drugs for treating cardiovascular d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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