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B group fumonisin degrading enzyme, its construction method and application

A fumonisin and enzyme-degrading technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of restricting popularization and application, food quality damage and toxic substances, etc.

Active Publication Date: 2022-05-31
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to physical, chemical and microbial degradation methods, it is easy to cause damage to food quality or introduce new toxic substances, which limits its popularization and application

Method used

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  • B group fumonisin degrading enzyme, its construction method and application
  • B group fumonisin degrading enzyme, its construction method and application
  • B group fumonisin degrading enzyme, its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1. Degradation of FBs by FumD protein

[0100] The human normal gastric epithelial cell line (GES-1) was used as the toxicity evaluation model to evaluate the cytotoxicity of the products after the individual reaction of FB1 and FumD proteins. After GES-1 cells were treated for 48 hours, the changes in cell viability were detected using CCK-8 kit (Japan Dojin Chemical Co., Ltd.).

[0101] The results showed that FB1 could significantly reduce cell activity, however, after FumD protein treatment, although the cytotoxicity of FB1 was alleviated, its degradation products were still toxic ( figure 1 ).

[0102] This phenomenon shows that further research is needed on better detoxification reagents and detoxification methods for Group B fumonisins.

Embodiment 2

[0103] Example 2. Construction and transformation of gene cloning of degrading enzyme FUMDI and eukaryotic expression vector

[0104] 1. Cloning of FumDI encoding gene

[0105]First, the inventors optimized the sequences of fumd gene (SEQ ID NO: 1) and fumi gene (SEQ ID NO: 3), respectively. After repeated research, analysis and experiments, their coding sequences were optimized respectively, and the optimized fumd gene sequences (SEQ ID NO: 2) and fumi gene sequences (SEQ ID NO: 4) were obtained respectively.

[0106] Secondly, the inventors used the optimized fumd and fumi as amplification templates, respectively, and the high-fidelity enzyme The target gene fragment was amplified by Max DNA Polymerase (purchased from Takara, Japan).

[0107] Amplification primers for fumd:

[0108] Forward primer fumd-F: 5'-gcgaattcatgcatcatcaccatcaccataaagaacaccaatgtaga-3' (SEQ ID NO:7);

[0109] Reverse primer fumd-R: 5'-gtctcgtaccattggccttagatggttgacatgctt-3' (SEQ ID NO:8);

[0110...

Embodiment 3

[0119] Embodiment 3, the expression of FUMDI protein of the present invention

[0120] Induced expression of protein: pick GS115 / pPIC9K-fumdi monoclone into 10ml YPED liquid medium, culture at 28°C, 220rpm / min for 8-12h, then insert into fresh BMGY liquid culture at 1:20 inoculum size , cultivated at 28°C, 220rpm / min to bacterial concentration OD 600 After reaching 1.0-1.5, all were inoculated into fresh BMMY liquid medium (containing 1% methanol), induced at 28°C, 220rpm / min for 96h (supplemented with 1% methanol every 24h), and the culture system was subjected to 4°C 4000rpm / min centrifugation to harvest the supernatant, which is the crude enzyme solution ( figure 2 B).

[0121] The collected crude enzyme solution was separated by SDS-PAGE electrophoresis, and stained with Coomassie brilliant blue R-250. After destaining, it was analyzed whether the protein band contained the target protein.

[0122] The results showed that the FUMDI protein achieved soluble expression ...

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Abstract

The invention provides a group B fumonisin degrading enzyme, its construction method and application. The present invention discloses a fumonisin degrading enzyme FUMDI for the first time, which can be effectively expressed, can form a correct protein space structure after expression, has good biological activity, and can efficiently degrade various B-group fumonisin compounds , greatly reducing the toxicity of the compound. The FUMDI of the present invention can not only degrade the prototype fumonisin, but also has a good degradation effect on degrading the hidden fumonisin (HFB1), and has a wider application range.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and in particular relates to a group B fumonisin degrading enzyme, a construction method and application thereof; the degrading enzyme of the invention can degrade group B fumonisins, and its degradation products Much less toxic than the prototype toxin. Background technique [0002] Fumonisin is a water-soluble metabolite mainly produced by F. verticillioides (F. verticillioides) and F. proliferatum (F. proliferatum). The linear diester compounds are divided into four categories: A, B, C, and P, and their prototypes are modified into concealed fumonisins through hydrolysis and other effects; the fumonisins that pollute commercial crops in nature are mainly FBs. The main types are divided into FB1, FB2 and FB3, among which FB1 is the most polluted and most toxic fumonisin. Fumonisin mainly contaminates corn and its products worldwide, especially in parts of Ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N9/10C07K19/00C12N15/62C12N15/81C12N1/19A23L5/20C12R1/84
CPCC12N9/18C12N9/1096C12N15/815A23L5/25C12Y301/01001C12Y206/01C07K2319/00
Inventor 武爱波于松余佃贞刘娜
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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