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Multifunctional enzyme for degrading mycotoxin and its application

A mycotoxin and hydrolase technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of toxic joint action and strong toxicity

Active Publication Date: 2022-06-17
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

[0006] Nowadays, it is known that effective degrading strains or degrading enzymes are mostly used for a single toxin, and there are few reports on biological agents such as strains that degrade two or more mycotoxins at the same time
In practice, multiple mycotoxins are often combined to contaminate food and feed, which may have a toxic joint effect, resulting in stronger toxicity

Method used

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  • Multifunctional enzyme for degrading mycotoxin and its application
  • Multifunctional enzyme for degrading mycotoxin and its application
  • Multifunctional enzyme for degrading mycotoxin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1. Construction and transformation of ZHDCP encoding gene cloning and eukaryotic expression vector

[0117] 1. Cloning of ZHDCP-encoding gene

[0118] The zhd and cp gene sequences are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively. In order to optimize the expression, the inventors conducted repeated experiments and designed codon-optimized sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0119] Further, in order to optimize the expression, the inventors modified the coding sequence of the CP protein, and truncated the coding sequence coding for amino acids 2-31 of the N-terminal of the CP protein. At the same time, in order to avoid the premature end of coding, the terminator TAA of the zhd coding gene was removed.

[0120] Using optimized zhd and cp as amplification templates, high-fidelity enzyme The target gene fragment was amplified by Max DNA Polymerase (purchased from Takara, Japan).

[0121] The amplification primers for Zhd...

Embodiment 2

[0133] Example 2. ZHDCP enzyme protein fermentation

[0134] 1. Preparation for Fermentation (Day 1)

[0135] Seed medium: 200mL YPD medium

[0136] Glycerol feed medium: 200 mL of 50% glycerol

[0137] Defoamers (polyethers)

[0138] Fermentation broth: 2L BMGY medium

[0139] Add 500 μl of antifoaming agent dropwise to the fermentation broth and pour it into the tank together for off-site sterilization. Calibrate the pH electrode before sterilization. After sterilization, connect the electrodes and pipelines, adjust the dissolved oxygen to zero, add 4mL of 500X biotin and 200mL of 10XYNB when the temperature is lowered to 30°C, adjust the speed to 50rpm, and adjust the air flowmeter to 2L / min overnight.

[0140] The seed solution was cultured at 16:30, and a single colony was picked and cultured in a 50 mL bottle of YPD medium (4 bottles in total) on a shaker at 28°C and 200 rpm.

[0141] 2. Fermentation on the tank (the next day)

[0142] 1. Initial Glycerol Culture ...

Embodiment 3

[0151] Example 3. Degradation of ZEN by recombinase ZHDCP

[0152] Experimental group: add 15μg ZEN and 5μg recombinase ZHDCP to a 1.5mL EP tube, dilute to 1mL with buffer (Tris-HCl buffer, containing 150mmol / L NaCl: 25mmol / L, pH=7.0), shake and mix well .

[0153] Control group: add 15 μg ZEN to a 1.5 mL EP tube, dilute to 1 mL with buffer (Tris-HCl buffer, containing 150 mmol / L NaCl: 25 mmol / L, pH=7.0), shake and mix well.

[0154] The control group and the experimental group were placed in a water bath at 35°C for 2 hours. After the reaction was completed, the centrifuge tube was placed in a water bath at 98°C for 2 minutes to inactivate, taken out and cooled to room temperature, and filtered with a water-based filter membrane.

[0155] The standard curve was prepared with first-grade water in the control group, and the standard concentration was 20, 50, 100, 200, and 500 ng / mL. The solution in the experimental group was appropriately diluted with first-grade water, and ZE...

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Abstract

The invention provides a multifunctional enzyme for degrading mycotoxin and its application. The present invention discloses a multifunctional enzyme ZHDCP for the first time, which can degrade more than one mycotoxin, can convert the toxin ZEN and OTA into low-toxic products respectively, the gene product is clear, and the toxicity is significantly lower than that of the prototype. The preparation of the ZHDCP protein of the invention is simple, can be soluble expressed and secreted into the culture medium, facilitates protein collection, and is suitable for large-scale production.

Description

technical field [0001] The present invention belongs to the technical field of genetic engineering and enzyme engineering; more particularly, the present invention relates to a multifunctional enzyme capable of degrading mycotoxins, a preparation method and application thereof, and the multifunctional enzyme can simultaneously degrade zearalenone and ochratoxin. Background technique [0002] Mycotoxins are a class of small-molecule toxic secondary metabolites produced by fungi (Aspergillus, Penicillium, Fusarium, etc.) under certain conditions that are harmful to animals and plants. These toxins mainly include deoxynivalenol (DON), zearalenone (ZEN), aflatoxins (AFs), ochratoxins and fumonisins , FBs) and so on. The contamination of agricultural products and food by mycotoxins is a global problem. According to the United Nations Food and Agriculture Organization (FAO), about 25% of the food supply is contaminated with mycotoxins, and 2% of the food is inedible due to the c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N9/48C12N15/62C12N15/81C12N1/19A23L5/20C12R1/84
CPCC12N9/18C12N9/485C12N15/815A23L5/25C07K2319/00
Inventor 武爱波余佃贞李凯琳刘娜
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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