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Amidase and coding gene, recombinant carrier, recombinant strain and application thereof

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

Active Publication Date: 2020-07-10
江苏奥迈生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the degradation efficiency of the existing enzymes used to degrade ochratoxin is not high, or not suitable for industrial applications. At present, there is no amidase with high degradation rate of ochratoxin and suitable for industrial production.

Method used

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  • Amidase and coding gene, recombinant carrier, recombinant strain and application thereof
  • Amidase and coding gene, recombinant carrier, recombinant strain and application thereof
  • Amidase and coding gene, recombinant carrier, recombinant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: cDNA synthesis and cloning of amidase gene

[0040] The strain is derived from the Stenotrophomonas bacteria obtained in the laboratory in the early stage. Through gene sequence determination, the open reading frame nucleotide sequence of the amidase gene was analyzed, and the upstream primer (adh117-F )(SEQ ID NO.3): 5'-CGC GGATCC ATGCCGATCCGCCGCCGC-3'; downstream primer (adh117-R) (SEQ ID NO.4): 5'-CCG CTCGAG TCACTGCTTGTAGATCACCCG-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, italic sequence It is the homologous sequence at the downstream end of the pGEX-4T-1 vector). Underlined, the sequence in italics is the homologous sequence at the downstream end of the pGEX-4T-1 vector), the amidase gene encoding the amino acid shown in SEQ ID NO.1 was obtained by in vitro amplification...

Embodiment 2

[0041] Example 2: Heterologous Expression of Stenotrophomonas Amidase Gene

[0042] The E. Coil BL21(DE3) / pGEX-4T-1 / adh117 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 weight (i...

Embodiment 3

[0043] Example 3: Application of Heterologous Recombination Detoxification Enzyme in Ochratoxin Degradation

[0044] 1. Experimental materials

[0045] The enzyme preparation is the crude enzyme solution of the crushed supernatant after the expression of the pGEX-4T-1 / adh239 transformant, and other reagents are analytically pure chemical reagents.

[0046] 2. Experimental method

[0047] Take 10 μL of the crushed supernatant after expression of the pGEX-4T-1 / adh117 transformant and put 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 provides amidase and a coding gene, recombinant carrier, recombinant strain and application thereof, and belongs to the technical field of enzyme engineering. The invention provides theamidase and the coding gene thereof, and also provides appropriate pH of the amidase and an application of the amidase to hydrolysis of ochratoxin and detoxifying of ochratoxin of foods. Under the room temperature condition, the amidase is used for treating ochratoxin A in a buffer system of which the pH is 7.3 for 2 minutes, the degradation rate of the ochratoxin A achieves 100%, and the degradation efficiency is unprecedented in the field.

Description

technical field [0001] The invention relates to an amidase and its coding gene, a recombinant vector, a recombinant bacterium and its 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...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/70C12N1/21A23L5/20
CPCA23L5/25C12N9/80C12N15/70
Inventor 周育陈楠王旭杜郑君
Owner 江苏奥迈生物科技有限公司
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