Fumonisin-degrading enzyme fumdsb and its gene and application

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

Active Publication Date: 2021-07-20
TIANJIN UNIV OF SCI & TECH
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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 hazards. In addition, the European Union also prohibits the use of chemical methods to eliminate fungi. toxin
Physical methods can remove a certain amount of FB1, but the requirements are high, and it is easy to change the quality and flavor of food

Method used

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  • Fumonisin-degrading enzyme fumdsb and its gene and application
  • Fumonisin-degrading enzyme fumdsb and its gene and application
  • Fumonisin-degrading enzyme fumdsb and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Obtaining Fumonisin Degrading Enzyme FumDSB Gene

[0039] The gene fragment derived from Sphingomonadales Bacterium fumonisin degrading enzyme FumDSB 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

[0040] Example 2 Preparation of Fumonisin Degrading Enzyme FumDSB

[0041] The expression vector pET28a was subjected to double enzyme digestion (EcoR I+Xho I), and at the same time, the gene FumDSB encoding mature fumonisin degrading enzyme was double enzyme digested (EcoR I+Xho I), and the gene fragment encoding the degrading enzyme and the expression vector were cut out The pET28a was ligated to obtain the recombinant plasmid pET28a-FumDSB containing the degrading enzyme FumDSB and transformed into Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli strain BL21(DE3) / FumDSB.

[0042] The recombinant expression vector of fumonisin degrading enzyme gene containing signal peptide sequence was constructed in the same way.

[0043] 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, i...

Embodiment 3

[0044] Example 3 Determination of the properties of fumonisin degrading enzyme FumDSB

[0045] The enzyme activity of fumonisin degrading enzyme is detected by high performance liquid chromatography, and the specific method is as follows:

[0046] (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.

[0047] (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.

[0048] (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 field of agricultural biotechnology, and in particular relates to fumonisin degrading enzyme FumDSB and its gene and application. The invention provides a fumonisin-degrading enzyme FumDSB from Sphingomonadales Bacterium, the amino acid sequence of which is shown in SEQ ID NO.1, and the invention provides a coding gene encoding the above-mentioned detoxification enzyme. The fumonisin degrading enzyme of the invention can be applied to industries such as agriculture, feed and food, and reduces the harm of fumonisin to animal and human health.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to fumonisin degrading enzyme FumDSB 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21A62D3/02A62D101/28C12R1/19
CPCA62D3/02A62D2101/28C12N9/18C12N15/70
Inventor 李中媛张同存赵军旗刘会会
Owner TIANJIN UNIV OF SCI & TECH
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