Fumonisin degrading enzyme FumDXA as well as gene and application thereof

A fumonisin and enzyme-degrading technology, applied in the field of agricultural biology, can solve the problems of changing food quality and flavor, high requirements, and large limitations of chemical methods, and achieve the effect of reducing harm

Active Publication Date: 2019-02-15
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of physical and chemical methods can remove fumonisins. However, chemical methods have relatively large limitations. Although they can reduce the content of FB1, they will introduce new chemical substances and increase safety haz

Method used

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  • Fumonisin degrading enzyme FumDXA as well as gene and application thereof
  • Fumonisin degrading enzyme FumDXA as well as gene and application thereof
  • Fumonisin degrading enzyme FumDXA as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Cloning of Fumonisin Degrading Enzyme FumDXA

[0036] The gene fragment of fumonisin degrading enzyme FumDXA derived from Xenophilus azovoran was obtained by artificial chemical synthesis, and the endonuclease site EcoR I was introduced at the 5' end, and the endonuclease site Xho I was introduced at the 3' end.

Embodiment 2

[0037] Example 2 Preparation of Recombinant Fumonisin Degrading Enzyme FumDXA

[0038] The expression vector pET28a was subjected to double enzyme digestion (EcoR I+Xho I), and at the same time, the gene FumDXA encoding fumonisin detoxification enzyme was double enzyme digested (EcoR I+Xho I), and the gene fragment encoding the detoxification enzyme was cut out and the expression vector pET28a After connection, the recombinant plasmid pET28a-FumDXA containing the degrading enzyme FumDXA was obtained and transformed into Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli strain BL21(DE3) / FumDXA.

[0039] Take the BL21(DE3) strain containing the recombinant plasmid, inoculate it in 100mL LB culture medium, shake it at 220rpm at 37℃ for 2-3h, and the OD 600 When the temperature is 0.6-0.8, add IPTG with a final concentration of 1 mM, induce for 20 hours at 25°C, and after the induction is over, collect the cells by centrifugation at 4°C. The supernatant was co...

Embodiment 3

[0040] Example 3 Determination of the properties of fumonisin degrading enzyme FumDXA

[0041] The enzyme activity of fumonisin degrading enzyme is detected by high performance liquid chromatography, and the specific method is as follows:

[0042] (1) FB1 standard stock solution: Weigh 1mg of the standard product, dissolve it in 10ml of acetonitrile and water (1:1), prepare a standard solution with a concentration of 100ug / mL, and store at -20°C.

[0043] (2) Sample preparation: Take 900ul of purified fumonisin degrading enzyme enzyme solution, add 100ul of FB1 standard stock solution to make the final concentration of FB1 10ug / ml, incubate at 37°C, 220rpm, in the dark for 20min.

[0044] (3) Sample derivatization: take 100 ul of the sample to be tested, add 400 ul of 50% acetonitrile water, 500 ul of OPA derivatization solution, mix for 30 s, inject the sample within 2 min of derivatization, and filter the membrane for testing. The enzymatic activity of the fumonisin degradi...

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Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to Fumonisin degrading enzyme FumDXA as well as a gene and an application thereof. The Fumonisin degrading enzyme protein FumDXA derived from Xenophilus azovorans is provided, the amino acid sequence of the Fumonisin degrading enzyme protein FumDXA is represented as SEQ ID NO.1, and the encoding gene forencoding the Fumonisin degrading enzyme is also provided. The Fumonisin degrading enzyme FumDXA has activity of degrading Fumonisin B1 and can be applied to agriculture, feed and food industries, sothat harm of Fumonisin to health of animals and humans is reduced.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to fumonisin degrading enzyme FumDXA and its gene and application. Background technique [0002] Fumonisin (FB) is some water-soluble secondary metabolites produced by Fusarium spp. under specific conditions. Fumonisins are diester compounds composed of polyhydric alcohols and tricarboxylic acids. The main active functional groups in the structure are primary amines, tricarboxylic acids, hydroxyl groups and aliphatic skeletons, which are closely related to their toxic effects. Studies have shown that fumonisins can cause kidney tumors and liver cancer in rats, equine leukomalacia (ELEM), porcine pulmonary edema (PPE), etc., and are closely related to the pathogenesis of human esophageal cancer. So far, 28 analogues of fumonisins have been found, which are divided into four classes, namely FA, ​​FB, FC and FP. In the B series, the most commonly found are fumonis...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21A23L5/20
CPCA23L5/25C12N9/18C12N15/70
Inventor 李中媛张同存赵军旗刘会会
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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