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Glyphosate oxidase mutant, cloning, expression and applications thereof

A technology of glyphosate and oxidase, applied in the field of genetic engineering and molecular biology, can solve the problems of insufficient degradation performance of glyphosate

Active Publication Date: 2020-04-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] For this reason, the technical problem to be solved by the present invention is to provide a glyphosate oxidase mutant to solve the problem of insufficient degradation performance of glyphosate oxidase to glyphosate in the prior art;

Method used

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  • Glyphosate oxidase mutant, cloning, expression and applications thereof
  • Glyphosate oxidase mutant, cloning, expression and applications thereof
  • Glyphosate oxidase mutant, cloning, expression and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Utilizes DNA shuffling technology to construct mutant library

[0038] In this example, the above-mentioned mutants B4S4, B4S6, B4S7, B4S9, and B4S11 were recombined to construct a library of glyphosate oxidase mutants, and the glyphosate oxidase mutant library was screened, and the affinity and catalytic efficiency for glyphosate were greatly improved. mutant.

[0039] The above recombinant plasmids pGEX-6p-B4S4, pGEX-6P-B4S6, pGEX-6p-B4S7, pGEX-6p-B4S9 and pGEX-6p-B4S11 were used as mutation templates respectively (Yao, Lin et al.2015), refer to Stemmer report The DNA shuffling method and the use of ultrasonic method to process DNA fragments, DNA shuffling (Stemmer 1994, Miller, Pislaru et al. 2002), and the formed recombinant plasmid transformed Escherichia coli (E. Further improvement of Bacillus glycine oxidase to obtain glyphosate oxidase with better glyphosate degradation activity and form a mutant library.

[0040] (1) Amplification of the template g...

Embodiment 2

[0070] Screening of embodiment 2 mutants

[0071] The method in this example refers to the screening method reported by (Zhan, Zhang et al. 2013).

[0072] Use a sterilized toothpick to pick the control bacteria (Escherichia coli DH5α containing the recombinant plasmid pGEX-6p-B4S7) and the clones in the mutant library and inoculate them into 600 μL liquid LB medium (add ampicillin to a concentration of 100 μg / ml) in a 96-deep-well plate, and at the same time store the bacteria in each well on an ampicillin-resistant plate as a backup. Seal the 96-deep-well plate, shake and culture at 37°C and 200rpm for 16 hours, add LB solution containing ampicillin (100 μg / ml), IPTG (final concentration 0.1 mM) and T7 bacteriophage (Tarahovsky, Ivanitsky et al.1994) to each well 200 μL of culture medium, 28°C, 200 rpm shaking culture for 6 hours, so that the protein was induced to express and lysed by phage and released; pipette 159 μL of crude enzyme solution after lysis (the supernatant...

Embodiment 3

[0079] Embodiment 3 mutant protein expression and purification

[0080] Recombinant plasmid pGEX-6p-B5R18 containing mutant B5R18 and B5R26 genes (see attached Figure 8 shown), pGEX-6p-B5R26 (see attached Figure 9 shown) were mixed with E.coli BL21(DE3) competent cells, transformed by electric shock, spread on ampicillin-resistant plates (ampicillin concentration 100 μg / mL), and cultured overnight at 37°C until a single colony visible to the naked eye grew. Pick a single clone and inoculate it into 20 mL of liquid LB medium (containing 100 μg / mL of ampicillin), and culture overnight at 37° C. with shaking at 200 rpm. Transfer the bacterial solution to 2L of fresh liquid LB medium (containing ampicillin 100 μg / mL) according to the amount of 1%, and culture at 37°C and 200rpm to OD 600 was 0.6, added the inducer IPTG with a final concentration of 0.1 mM, induced and cultured for 12 hours at 22°C and 160 rpm to express the protein. The bacteria were collected by centrifugati...

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Abstract

The invention belongs to the technical field of gene engineering and molecular biology, specifically relates to a glyphosate oxidase mutant artificially modified by a directed evolution technology andcapable of remarkably improving glyphosate degradation performance, and further discloses applications of the mutant and a coding gene thereof in the fields of cultivation of novel glyphosate-resistant crops and biodegradation of glyphosate pollution. According to the scheme, a glyphosate oxidase mutant library is constructed on the basis of known glyphosate oxidase mutants through a DNA shuffling technology, and two mutants B5R18 and B5R26 with improved glyphosate catalytic efficiency are screened from a large number of mutants, and have high glyphosate degradation activity, so that the defect of low activity of existing glyphosate oxidase is effectively solved, and the mutant has great potential application value in the fields of glyphosate-resistant crop cultivation, degradation treatment of glyphosate pollution and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and molecular biology, and specifically relates to a glyphosate oxidase mutant artificially transformed by directed evolution technology that can significantly improve the degradation performance of glyphosate, and further discloses the mutant and its coding The application of genes in the cultivation of new glyphosate-resistant crops and the biodegradation of glyphosate pollution. Background technique [0002] Glyphosate (trade name Roundup) chemical name N-phosphonomethylglycine (N-(phosphonomethyl) glycine, GLP), its molecular formula is C 3 h 8 NO 5 P, with a relative molecular weight of 169.1, glyphosate has a similar structure to glycine and is a derivative of glycine. Glyphosate is a white solid, non-volatile, with a high melting point (200°C), good stability, and low solubility in water (1.2%, 25°C). Glyphosate is a non-selective, high-efficiency herbicide that is widely us...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C40B50/06C40B40/08C12R1/19
CPCC12N9/0022C12N15/70C12N15/8275C12Y104/03019C40B40/08C40B50/06
Inventor 刘子铎吴高兵林拥军郭一鸣
Owner HUAZHONG AGRI UNIV
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