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Phosphotriesterase mutant as well as preparation method and application thereof

A phosphotriesterase and mutant technology, applied in the biological field, can solve problems such as toxic and side effects, and achieve the effect of improving hydrolysis activity

Inactive Publication Date: 2012-11-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Organophosphorus compounds have strong toxic and side effects on humans and livestock

Method used

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  • Phosphotriesterase mutant as well as preparation method and application thereof
  • Phosphotriesterase mutant as well as preparation method and application thereof
  • Phosphotriesterase mutant as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Cloning of wild-type phosphotriesterase gene

[0019] (1) Cultivation of thermophilic bacterium Geobacillus kaustophilus HTA426 and extraction of genomic DNA

[0020] Resuscitate and cultivate according to the culture method provided by the Japan Collection of Microorganisms, first prepare Rehydration fluid 7: 5g / L peptone (peptone), 3g / L beef extract (beef extract), 10g / LNaCl, dilute to volume with deionized water, Adjust the pH to 7.0, and sterilize under high temperature and high pressure.

[0021] Add 0.5ml rehydration fluid to the lyophilized powder of Geobacillus kaustophilus HTA426 cells, dissolve and transfer to a test tube containing 5ml LB medium, culture at 60°C with shaking at 180rpm for 24 hours. Then transfer to a Erlenmeyer flask filled with 100ml LB medium and culture at 60°C for 48 hours.

[0022] Take 5ml of bacterial culture to collect the bacteria, resuspend the pellet with 200μL of 25mmol / L Tris-HCl buffer (pH8.0, containing 50mmol / L gl...

Embodiment 2

[0031] Embodiment 2: Utilize the method of error-prone PCR to construct random mutation library

[0032] Random nucleotide mutations were introduced into the phosphotriesterase gene of G. kaustophilus HTA426 by error-prone PCR. The primers used were the same as those used for cloning the phosphotriesterase gene in Example 1. The error-prone PCR system is as follows: 2 μl Taq polymerase (TaKaRa company), 10 μl 10× buffer (without Mg) in 100 μl reaction system 2+ ), 8 μl dCTP (10 mM), 8 μl dTTP (10 mM), 8 μl dNTP mixture (each nucleotide concentration 2.5 mM), 24 μl MgCl 2 (25mM), 2μl MnCl 2 (10 mM), 1 μl pET28a recombinant plasmid carrying the phosphotriesterase gene, 2 μl upstream primer (20 pmol / μl), 2 μl downstream primer (20 pmol / μl), 33 μl ultrapure water. Error-prone PCR reaction conditions are the same as those described in Example 1. Error-prone PCR products were detected by 1% agarose gel electrophoresis after the reaction.

[0033] After the error-prone PCR ampli...

Embodiment 3

[0034] Example 3: Screening of Mutants

[0035] Pick clones from the mutant library with sterilized toothpicks, and inoculate them into 96-well cell culture plates containing 200 μl of LB liquid medium containing 50 ug / ml kanamycin in each well, and each well corresponds to each specific transformant. At the same time, wild-type clones and empty bacteria were inoculated in 96-well cell culture plates as positive and negative controls, respectively. 37°C, 160rpm, and culture on a shaker for about 16h, so that the bacterial growth reaches the plateau stage. Add 50% v / v sterile glycerol to each well to a final glycerol concentration of 20%, and freeze at -80°C.

[0036] Take 5 μl of bacterial solution from the bacterial well of the original bacterial plate and transfer it to 150 μl LB liquid medium (containing 50ug / ml kanamycin, 1mM Co 2+ ) in the corresponding wells of the 96-well culture plate. The replica plate was grown to OD at 37°C with careful shaking at 180rpm 600 It ...

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Abstract

The invention provides a phosphotriesterase mutant as well as a preparation method and application of the phosphotriesterase mutant. According to the invention, a mutant with phosphotriesterase activity markedly improved is finally obtained by starting from a phosphotriesterase gene which is from Geobacillus kaustophilus HTA426 of thermophilic bacteria and then carrying out rounds of mutation and screening through an error-prone PCR (Polymerase Chain Reaction) method; and the mutations involved in amino acid sequence of the mutant are Phe28Ile, Tyr99Leu, Thr171Ser, Phe228Leu, Asn269Ser, Val270Gly, Trp271Cys and Gly273Asp. Compared with common organic phosphorous insecticides, the wild type specific activity of the mutant is markedly improved and the mutant has wide application prospect in the field of biodegradation of organic phosphorous poisions.

Description

technical field [0001] The invention relates to biotechnology, in particular to a phosphotriesterase mutant and its preparation method and application. Background technique [0002] Phosphotriesterase (Phosphotriesterase, EC 3.1.8.1) can hydrolyze a broad spectrum of organophosphorus compounds, which are widely used in agricultural pesticides and chemical warfare nerve agents. ester bond to detoxify it. As shown in the following formula: [0003] [0004] Organophosphorus compounds have extremely strong toxic and side effects on humans and livestock. Some of the more common organophosphorus compounds and their toxicity are listed in Table 1. Up to now, more than 100 kinds of organophosphorus pesticides have been widely used all over the world, accounting for about 38% of the kinds of pesticides. In the United States alone, about 50,000 tons of organophosphorus pesticides are used in agricultural production each year, in addition to about 400,000 liters of coumaphos wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N1/21A62D3/02C12R1/19A62D101/04A62D101/28
Inventor 冯雁张宇安娇杨广宇
Owner SHANGHAI JIAO TONG UNIV
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