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Ochratoxin detoxification enzyme, encoding gene, recombinant vector and application thereof

A technology of ochratoxin and recombinant vector, applied in the field of enzyme engineering, can solve the problems of unsuitability for industrial application, low degradation efficiency of ochratoxin, etc.

Active Publication Date: 2020-07-10
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing enzymes for degrading ochratoxin have low degradation efficiency, or are not suitable for industrial applications. At present, there is no enzyme with high degradation rate of ochratoxin and suitable for industrial production.

Method used

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  • Ochratoxin detoxification enzyme, encoding gene, recombinant vector and application thereof
  • Ochratoxin detoxification enzyme, encoding gene, recombinant vector and application thereof
  • Ochratoxin detoxification enzyme, encoding gene, recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: cDNA synthesis and cloning of ochratoxin detoxification enzyme gene

[0046] The strain is derived from the lysobacterium strain of Xanthomonas bacteria obtained in the early stage of the laboratory. Through gene sequence determination, the nucleotide sequence of the open reading frame of the ochratoxin A hydrolase gene was analyzed, and the complete coding reading frame was designed and amplified. Primer upstream primer (adh239-F) (SEQ ID NO.3): 5'-ATCTGGTTCCGCGT GGATCC ATGACCGTCCGCCTGGTCCG-3'; downstream primer (adh239-R) (SEQ ID NO.4): 5'-TCACGATGCGGCCG CTCGAG TCATGGCGACGTCCCGGGC-3', respectively introduce restriction endonuclease sites on the upstream and downstream primers (depending on the carrier selected, BamHI and XhoI enzyme cutting sites have been added in the present invention, underlined, italicized sequence It is the homologous sequence at the downstream end of the pGEX-4T-1 vector). It has been marked with an underline, and the sequence in ...

Embodiment 2

[0047] Example 2: Heterologous Expression of Ochratoxin Detoxification Enzyme Gene

[0048] The E. Coil BL21(DE3) / pGEX-4T-1 / adh239 transformant obtained in Example 1 was shaken overnight in 100 mL LB liquid medium containing 100 μg / mL ampicillin. Draw 1.0 mL of seed bacteria liquid and inoculate it into fresh 100 mL of LB liquid medium (containing 100 μg / mL ampicillin), and culture at 37° C. with shaking at 180 r / min. When the bacteria solution OD 600 When it reaches 0.6, add 0.2mmol / mL IPTG to induce expression at 16°C for 4h. Refrigerate and centrifuge at 7000r / min for 10min, discard the supernatant. Suspend the cells with 10 mL of 1×phosphate buffered saline (PBS), and disrupt the cells by ultrasonic in an ice bath. Centrifuge and refold the pellet for inclusion body renaturation. The results of SDS-PAGE electrophoresis show that the enzyme is present in the supernatant and the precipitate (inclusion body) after the bacterial cell is broken, and its apparent molecular w...

Embodiment 3

[0049] Example 3: Application of Heterologous Recombinant Ochratoxin Detoxification Enzyme in Degradation of Ochratoxin

[0050] 1. Experimental materials

[0051] The enzyme preparation is the crude enzyme solution of the supernatant after pGEX-4T-1 / adh239 transformant expression, and other reagents are analytically pure chemical reagents.

[0052] 2. Experimental method

[0053] Take 10 μL of the pGEX-4T-1 / adh239 transformed supernatant after expression and place it in a 2 mL centrifuge tube, add it to 490 μL of ochratoxin A standard buffer (use 1*PBS to prepare pH 7.3), ochratoxin A The final concentration of the solution is about 40 μg / L, react for 2 minutes, and add 1.5 mL of acetonitrile to the reaction system to stop the reaction. High performance liquid chromatography was used to detect the residual amount of OTA. The experimental results showed that after 2 minutes of reaction, the broken supernatant degraded all OTA.

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Abstract

The invention discloses an ochratoxin detoxification enzyme, an encoding gene, a recombinant vector and an application thereof, and belongs to the technical field of enzyme engineering. The inventionprovides the ochratoxin detoxification enzyme and the encoding gene thereof, and also provides the optimum pH of the ochratoxin detoxification enzyme and the application of the ochratoxin detoxification enzyme in degrading ochratoxin and ochratoxin biological detoxification in food. When ochratoxin A is processed by the ochratoxin detoxification enzyme at a temperature of 37 DEG C and at the pH of7.3 for 2min in a buffer system, the degradation rate of ochratoxin A reaches 100%, and the degradation efficiency is unprecedented in the field.

Description

technical field [0001] The invention relates to an ochratoxin detoxification enzyme and its coding gene, recombinant vector and application, and belongs to the technical field of enzyme engineering. Background technique [0002] Aspergillus mycotoxins are mainly a class of secondary metabolites produced by toxin-producing Aspergillus strains such as Aspergillus flavus, Aspergillus parasiticus and ochratoxin. More than 20 structural analogues have been isolated and identified, among which aflatoxin B1 (AFB1) and Ochratoxin A (OTA) is the most toxic and most widely polluted in agricultural production and food industry. In 1993, the Cancer Research Institute of the World Health Organization (WHO) classified aflatoxin as a class I carcinogen and ochratoxin as a class IIB carcinogen. Studies have shown that ochratoxin is the main cause of human Balkan nephropathy. Because of its strong nephrotoxicity, as well as possible carcinogenicity, teratogenicity and mutagenicity, it has g...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55A23L5/20
CPCA23L5/25C12N9/14
Inventor 周育王旭陈楠杜郑君
Owner ANHUI AGRICULTURAL UNIVERSITY
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