Gene for coding aflatoxin degradation enzyme and method for obtaining high-efficiency aflatoxin degradation enzyme

A kind of aflatoxin and degrading enzyme technology, applied in feed, biotechnology application or agriculture, food field, can solve the problems of low degradation efficiency, harsh use conditions, low yield and activity of natural enzymes, etc.

Inactive Publication Date: 2014-02-05
HUAZHONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

In order to solve the problem of low yield and activity of the obtained natural enzyme, Liu's research group tried to express the detoxifying enzyme in prokaryotic and eukaryotic expression systems, but these enzymes still have low degradation efficiency, poor stability, and harsh use conditions, etc. Problems affecting the further application of ADTZ (Hu Rong, Liu Daling, Xie Chunfang, Yao Dongsheng. Soluble expression, purification and circular dichroism analysis of aflatoxin detoxification enzyme in Escherichia coli. China Biotechnology Journal. 2011, 31( 4), 71-76)

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  • Gene for coding aflatoxin degradation enzyme and method for obtaining high-efficiency aflatoxin degradation enzyme
  • Gene for coding aflatoxin degradation enzyme and method for obtaining high-efficiency aflatoxin degradation enzyme
  • Gene for coding aflatoxin degradation enzyme and method for obtaining high-efficiency aflatoxin degradation enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The optimized aflatoxin degrading enzyme gene of embodiment 1 ( Opt-ADTZ )

[0030] Search for the aflatoxin degrading enzyme gene (GeneBank No. AY941095.1) on NCBI, and optimize the codons to obtain the optimized aflatoxin degrading enzyme gene ( Opt-ADTZ ), the nucleotide sequence of which is shown in SEQ ID NO.1, and the sequence similarity between the optimized gene and the original gene is 78%. After the synthesis of Opt-ADTZ, it was cloned into the pUC19 vector to obtain pUC19-Opt-ADTZ.

Embodiment 2

[0031] Example 2 Construction of aflatoxin degrading enzyme expression vector pCold-II-Opt-ADTZ

[0032] (1) Extraction of plasmid

[0033] Take 2 Erlenmeyer flasks containing 20mL LB (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, ampicillin 30mg / L) medium, and put pUC19-Opt-ADTZ, pCold-II A single colony of the plasmid was added to the culture medium, cultured overnight at 37°C and 220 rpm, and the plasmid was extracted according to the SDS lysis method. After the plasmid extraction is completed, mark it and put it in a 4°C refrigerator for use in the next step.

[0034] (2) PCR amplification Opt-ADTZ , Opt-ADTZ Digestion with pCold-II

[0035] PCR amplification reaction system: plasmid (pUC19-Opt-ADTZ) 1 μL, 10×buffer 5 μL, dNTPs 4 μL, Opt-ADTZ upstream primer 1 μL, Opt-ADTZ downstream primer 1 μL, Taq DNA polymerase 1 μL, add ddH 2 0 to 50 μL.

[0036] Opt-ADTZ upstream primer is 5'-C GCATA TGC ATGGCGACCACCACC-3',

[0037] Opt-ADTZ downstream primer is 5'-AT ...

Embodiment 3

[0047] Example 3 Expression and purification of aflatoxin degrading enzyme

[0048](1) Optimization of induction conditions

[0049] The concentration of the bacterial solution before induction, the concentration of the inducer tetracycline and IPTG, the induction expression temperature, and the induction time were optimized

[0050] The pCold-II-Opt-ADTZ plasmid in Escherichia coli DH5α was extracted, and the extracted plasmid was transformed into BL21-PG-Tf2 competent medium. Activate the expressing bacteria containing the target gene in a 20 mL LB medium flask containing 30 mg / L ampicillin at 37 °C and 220 rpm for 16 hours, take the activated bacterial liquid and inoculate it in 500 mL LB medium at an inoculation amount of 2.5%. Choose different induction conditions to induce expression. After induction of expression, the bacteria were collected, and the expression of the target protein was detected by SDS-PAGE to determine the optimal induction conditions. Finally, th...

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Abstract

The invention discloses a gene for coding an aflatoxin degradation enzyme and a method for obtaining a high-efficiency aflatoxin degradation enzyme. The nucleotide sequence of the gene for coding the aflatoxin degradation enzyme is shown as SEQ ID NO. 1. The method for obtaining the high-efficiency aflatoxin degradation enzyme comprises the following steps: cloning the gene shown as the SEQ ID NO. 1 to a carrier pCold-II; converting to Escherichia coli BL21-PG-Tf2 to obtain expression thallus of the aflatoxin degradation enzyme; inducing expression of the expression thallus by using tetracycline and IPTG (isopropyl-beta-d-thiogalactoside); collecting the thallus; crushing; purifying by using a fusion protein expressed tag on the pCold-II to obtain the high-efficiency aflatoxin degradation enzyme. The purity of the obtained aflatoxin degradation enzyme reaches over 95 percent by optimizing the expression carrier and the expression condition, and the efficiency for degrading the aflatoxin B1 reaches 161.7 nmol / min / mg.pr.

Description

[0001] technical field [0002] The invention relates to the application of biotechnology or the fields of agriculture, food, feed and the like, in particular to a gene encoding an aflatoxin-degrading enzyme and a method for obtaining a high-efficiency aflatoxin-degrading enzyme. Background technique [0003] Aflatoxin (Aflatoxin, AFT) is a metabolite of fungi such as Aspergillus flavus and Aspergillus parasiticus, and is considered to be the most toxic, most widely affecting, and most stable type of mycotoxin found in nature. It is a common mycotoxin in crops Pollutants, widely polluting feed, grain and oil, animal and plant food, etc., have brought huge losses to agricultural production, brought great hidden dangers to food safety, and also seriously threatened public health. source". In recent years, due to the increase of global abnormal climate, global trade of raw materials, energy crisis and shortage of raw materials, AFT pollution has become increasingly serious, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/62C12R1/69
Inventor 冯玲玲万坚李俊金晶任彦亮黄培培冯江涛
Owner HUAZHONG NORMAL UNIV
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