Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent

A technology of compound enzyme preparation and recombinant strain, applied in the direction of non-surface active detergent composition, hydrolytic enzyme, detergent composition, etc., can solve the problems of low content, difficult to produce cheaply, etc., achieve simple process operation, reduce production cost effect

Inactive Publication Date: 2011-09-28
SHENZHEN SYDSCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low content of pesticide-degrading enzymes in natural raw enzyme beads, it is difficult to achieve industrialized and cheap production

Method used

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  • Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent
  • Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent
  • Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent

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Experimental program
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Effect test

Embodiment 1

[0040]The compound enzyme preparation process of the preferred embodiment of the present invention comprises the following steps: a), constructing the recombinant organophosphate hydrolase pET28-OPH, that is, applying gene recombination technology to clone the organophosphate hydrolase gene into the pET28 vector, and constructing the expression plasmid pET28-OPH , the plasmid has a T7 promoter, which can express recombinant organophosphate hydrolase protein under the induction of IPTG; b), prepare a recombinant organophosphate hydrolase expression strain with controlled cleavage; c), hydrolyze the obtained recombinant organophosphate The enzyme expression strain is subjected to engineering fermentation culture, and pET28-OPH is induced to express the lyase; d), the fermentation broth obtained in step c) is used to prepare a compound enzyme preparation. In this way, by carrying out engineering fermentation culture on the obtained recombinant organophosphate hydrolase expression ...

Embodiment 2

[0055] First use PCR to amplify the OPH gene of organophosphate hydrolase, and then design specific oligonucleotide primers according to the coding sequence of organophosphate hydrolase. (Restriction enzymes) NdeI and BamHI double-digested the pET15 vector, ligated with the OPH amplified product of the same digestion; transformed the ligated product into temperature-sensitive self-lysing host strain BL21 NTC4828, and screened on a 100mg / L ampicillin plate Positive clone.

[0056] Pick a single colony and inoculate it in liquid LB medium, and culture it in a shaker at 30°C and 150-200rpm for 10-15 hours; sterilize the fermenter at 121°C for 30 minutes, then insert 5% of the obtained seed liquid, and pass The dissolved oxygen value was controlled by rotation speed and ventilation. When the OD value in the tank reached 3, IPTG was added for induction for 16 hours, and then the temperature of the fermenter was increased to 42°C for 3 hours to induce E. coli to express lyase. Fina...

Embodiment 3

[0058] 1. Construction of pET28-OPH

[0059] Firstly, according to the coding sequence of organophosphorus hydrolase, specific oligonucleotide primers were designed, with NcoI and XhoI restriction sites at both ends, and the OPH gene was amplified by PCR. The primer sequences are as follows:

[0060] P1 5-AAACCCCCATGGATGAGCATTGGAACCGGCGATCGTA-3

[0061] P2 5-CCCAAACTCGAGGCTTGCCCGAAGAGTCGGGCTCAGA-3,

[0062] The pET28 vector was double-digested with restriction enzymes, ligated with the OPH amplified product of the same digestion, the ligated product was transformed into Escherichia coli BL21 (DE3), and positive clones were screened on a 50mg / L kanamycin plate.

[0063] 2. Construction of pET22-λSR

[0064] 1. Synthesis of the arabinose pBAD promoter

[0065] The pBAD promoter was artificially synthesized according to the nucleotide sequence of the arabinose pBAD promoter, and Bg1II and NcoI restriction sites were added to its two ends, respectively. The sequence compositi...

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Abstract

The invention relates to a recombinant strain expressing organophosphorus hydrolase, a complex enzyme preparation technology, a complex enzyme preparation and a fruit and vegetable detergent, wherein the recombinant strain comprises a controllable lyase gene. The complex enzyme preparation technology comprises the following steps: culturing the recombinant strain expressing organophosphorus hydrolase, adding proper start-up condition induced expression lyase; and using the obtained culture solution to prepare the complex enzyme preparation. The recombinant strain expressing organophosphorus hydrolase is cultured and the expression of lyase lysed cells is induced, thus the possible influences of complicated mechanical and chemical pyrolysis methods on the expression of enzyme activity can be avoided and the cheap industrialized production is convenient to realize. The complex enzyme preparation produced on the basis of the preparation technology has organophosphorus hydrolase and cell lyase simultaneously, has the double functions of pesticide degradation and sterilization and can be used in the development of the new fruit and vegetable detergent.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a recombinant bacterial strain expressing organophosphate hydrolase, a preparation process of a compound enzyme, a compound enzyme preparation and a fruit and vegetable detergent. Background technique [0002] Pesticides are necessary means of production in agricultural production. There are more than 14,000 types of chemical pesticides in the world, and the annual output is 3 million tons in terms of raw materials. my country is a big producer of highly toxic pesticides, among which the use of organophosphorus pesticides accounts for 57% of the total pesticides. Pesticide residues will cause serious pollution to the environment and food, and the pollution is widespread and lasts for a long time, seriously affecting people's health And the export of agricultural products has aroused widespread concern from more and more societies. [0003] How to effectively deal with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N1/19C12N1/21C12N15/55C12N15/56C12N15/60C12N15/63C12N9/16C12N9/36C12N9/88C11D7/42C11D3/386
Inventor 郭永超刘牧龙王艳平
Owner SHENZHEN SYDSCI TECH
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