Organophosphorus degrading enzyme mutants and coding genes and application thereof

A technology for degrading enzymes and organophosphorus, applied in the field of enzyme mutants, mutants of organophosphate degrading enzymes and their coding genes, and degrading organophosphorus pesticides, which can solve the problems of low sulfur and phosphorus degradation rates

Active Publication Date: 2010-12-15
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 organic phosphorus degrading enzyme OPHC2 (the accession number in GenBank is AJ605330): using the published MPH crystal structure derived from Pseudomonas sp.WBC-3 as a template, Accelrys Discovery Studio software (Ver.2.5) was used The software builds the three-dimensional structure of OPHC2 and determines the active center. Its three-dimensional structure is as follows figure 1 . OPHC2 is a homodimer, belonging to a typical β / α (TIM) folding barrel structure, in which 8 folding structures form a barrel, which is the active center region of the enzyme, and contains two Zn 2+ . According to the spatial structure characteristics of OPHC2, five mutation regions were designed at the enzyme-substrate binding pocket near the active center, and the mutations in these regions could not affect the structure of the catalytic active center of the enzyme. To ensure that the selected amino acid pairs can be close to ...

Embodiment 2

[0067] Embodiment 2 Obtaining of Mutant Gene Fragment

[0068] The technique of overlap extension PCR was used. Design a pair of flanking primers at both ends (a and d) and five pairs of primers (b and c) containing the desired mutation site, wherein the b primer contains the desired mutation site. The mutation site is a double-point saturation mutation, and the intermediate 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 sequences are shown in Table 1. Two rounds of PCR were carried out for each point mutation. In the first round of PCR, primers a and c were used to amplify the DNA fragment of the upstream sequence of the gene, and primers b and d were used to amplify the DNA fragment containing the mutation site and its downstream sequence. . Use agarose gel to recover these two pieces of DNA fragments, and then mix the two pieces of PCR products. Since b and c have overlappi...

Embodiment 3

[0076] The construction of embodiment 3 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 ligation system was: pUC19 2 μL, PCR product 6.5 μL, T 4 Ligase 0.5 μL, 10X reaction buffer 1 μL, mix gently, react overnight at 16°C. Each library makes 5 connection systems.

[0078] Transformation: Freeze and thaw Top10 competent cells stored at -70°C on ice, take 50 μL of competent cells and add them 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 90 sec, then immediately placed on ice for 2 min, and added 400 μL of pre-cooled LB to equilibrate to room temperature, and incubated at 200 rpm for 1 h at 37°C on a shaker. Add 4 μL of 1mol / L IPTG and 40 μL of 20 mg / ml X-gal to mix, and spread all the transformed bacterial solution o...

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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 invention relates to an enzyme mutant, in particular to a mutant of an organophosphorus degrading enzyme and its coding gene. The invention also relates to the application of the organophosphate degrading enzyme mutant in degrading organophosphorus pesticides, belonging to the field of organophosphate 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 types, of which more than 300 are commonly used, of which organophosphorus 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, pesticides have ...

Claims

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Application Information

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Patent Type & Authority Applications(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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