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Organophosphorus degrading enzyme mutants and coding genes and application thereof

A technology for degrading enzymes and organophosphorus, applied to mutants of organophosphorus-degrading enzymes and their encoding genes, applications in degrading organophosphorus pesticides, and the field of enzyme mutants, which can solve problems such as low degradation rate of sulfur and phosphorus

Active Publication Date: 2011-11-30
北京森根比亚生物工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] OPHC2 belongs to methyl parathion hydrolase, the most suitable substrate for its degradation is methyl parathion, and the degradation rate of ethyl parathion is low

Method used

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  • Organophosphorus degrading enzyme mutants and coding genes and application thereof
  • Organophosphorus degrading enzyme mutants and coding genes and application thereof
  • Organophosphorus degrading enzyme mutants and coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The first is to analyze the three-dimensional structure of the organophosphate degrading enzyme OPHC2 (the accession number in GenBank is AJ605330): using the published crystal structure of MPH derived from Pseudomonas sp.WBC-3 as a template, using Accelrys Discovery Studio software (Ver.2.5) The software constructs the three-dimensional structure of OPHC2 and determines the active center. The three-dimensional structure is as figure 1 . OPHC2 is a homodimer, belonging to a typical β / α (TIM) folding barrel structure, in which 8 folding structures constitute a barrel, which is the active center region of the enzyme, and contains two Zn in the middle. 2+ . According to the spatial structure characteristics of OPHC2, five mutation regions are designed in the pockets where the enzyme binds to the substrate near the active center. These mutations cannot affect the structure of the enzyme catalytic active center. To ensure that the selected amino acid pairs are close to each ...

Embodiment 2

[0067] Example 2 Obtaining mutant gene fragments

[0068] The overlap extension PCR technique is used. Design a pair of flanking primers (a and d) at both ends and five pairs of primers (b and c) containing the desired mutation sites, wherein the b primer contains the desired mutation site. The mutation site is a double-point saturation mutation. The middle primer of the amino acid pair to be mutated corresponds to 6 bases, and the base corresponding to each amino acid is replaced with nnk. The primer sequence is shown in Table 1. For each point mutation, two rounds of PCR were carried out. The first round of PCR used primers a and c to amplify the DNA fragments of the upstream sequence of the gene, while using primers b and d to amplify the DNA fragments containing the mutation site and its downstream sequence . Use an agarose gel to recover these two DNA fragments, and then mix the two PCR products. Because b and c have overlapping regions, the overlapping fragments anneal be...

Embodiment 3

[0076] Example 3 Construction of Saturation Mutant Library

[0077] Ligation: The PCR product recovered in Example 2 was directly digested with BamHI and HindIII and then ligated with the pUC19 vector treated with the same double digestion. The connection system was: pUC19 2μL, PCR product 6.5μL, T 4 Ligase 0.5μL, 10X reaction buffer 1μL, mix gently, and react at 16°C overnight. Each library does 5 connection systems.

[0078] Transformation: Freeze and thaw Top10 competent cells stored at -70°C on ice, add 50μL of competent cells to the above connection system, gently pipette to mix, and ice bath for 30 minutes. The ice-bathed system was accurately heat-shocked at 42°C for 90sec, and then immediately placed on ice for 2min, and 400μL of pre-cooled LB was added to equilibrate to room temperature, 200rpm, 37°C shaker and incubated for 1h. Add 4μL of 1mol / L IPTG and 40μL of 20mg / ml X-gal to mix, all the transformed bacteria solution is coated with LB (containing 100μg / mL, Amp) 23cm×...

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Abstract

The invention discloses organophosphorus degrading enzyme mutants and coding genes and application thereof. Site-directed mutation screening is performed on original enzyme coding genes to obtain three organophosphorus degrading enzyme mutants according to the spatial structure characteristic of organophosphorus degrading enzyme OPHC2 by computer-aid molecular design, and the amino acid sequences of the three mutants are shown by SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6 respectively. Compared with the original enzyme, the organophosphorus degrading enzyme mutants improve the ethyl-parathion degradation specific activities by 2.45 times, 2.56 times and 4.51 times, and the degradation capabilities (kcat / Km value) of the organophosphorus degrading enzyme mutants are 2.7 times, 3.3 times and 4.2 times that of the original enzyme. Enzymatic property analysis shows that the enzymatic property of the organophosphorus degrading enzyme mutants is not changed compared with the original enzyme, and the both have good heat resistance.

Description

Technical field [0001] The present invention relates to enzyme mutants, in particular to an organophosphorus degrading enzyme mutant and its coding gene. The present invention also relates to the application of the organophosphorus degrading enzyme mutant in degrading organophosphorus pesticides, belonging to the field of organophosphorus degrading enzymes. Background technique [0002] Organophosphorus pesticides are the most widely used pesticides in my country and even in the world. At present, the annual output of chemical pesticides in my country is more than 2 million tons, with a total of more than 600 kinds, of which more than 300 commonly used varieties, of which organic phosphorus pesticides account for more than 70%. The production of pesticides in my country not only meets the needs of domestic agricultural production, but also meets the needs of world agricultural production, and has made great contributions to the development of world agriculture. In my country, pe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A62D3/02A62D101/04A62D101/28
Inventor 伍宁丰范云六初晓宇田健吕红
Owner 北京森根比亚生物工程技术有限公司
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