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Aflatoxin degrading enzyme secreted by Bacillus subtilis, and applications thereof

A technology of Bacillus subtilis and aflatoxin, which is applied in the field of microbiology and genetic engineering, can solve the problems of Bacillus subtilis aflatoxin degrading enzyme, etc., and achieve the effect of specific action, high degradation efficiency and mild effect

Active Publication Date: 2018-03-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no research report on the aflatoxin-degrading enzyme of Bacillus subtilis and the full-length gene of the enzyme

Method used

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  • Aflatoxin degrading enzyme secreted by Bacillus subtilis, and applications thereof
  • Aflatoxin degrading enzyme secreted by Bacillus subtilis, and applications thereof
  • Aflatoxin degrading enzyme secreted by Bacillus subtilis, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 The acquisition and purification of Bacillus subtilis AF degrading enzyme BADE

[0031] 1. Preparation of Bacillus subtilis aflatoxin degrading enzyme (BADE)

[0032] Activation of seed solution: Inoculate 50 mL of LB medium with Bacillus subtilis ANSB060 strain preserved in 1 mL of milk powder, put it in a constant temperature shaking shaker at 37°C, culture at 180-200 r / min, cultivate for 24 hours, and activate twice. The LB medium is: tryptone 10g, sodium chloride 10g, yeast extract powder 5g, agar 16g (solid medium), 1000mL distilled water, adjust the pH value to 7.2, and sterilize by high pressure steam at 121°C for 20min.

[0033] Preparation of seed liquid: the seed liquid (1×10 9CFU / mL) was added to a 300mL Erlenmeyer flask containing 80mL of fermentation medium, placed in a constant temperature shaker at 37°C, 200r / min, and incubated for 24h.

[0034] Preparation of fermentation supernatant: 6 L of LB medium was loaded into a 10 L fermenter, and aft...

Embodiment 2

[0061] Example 2 Obtaining of the characteristic amino acid sequence of aflatoxin degrading enzyme BADE

[0062] The active protein was collected by SDS-PAGE electrophoresis to collect the target bands, and then carried out in-gel enzymatic hydrolysis, and the enzymatic peptides were extracted and separated by capillary liquid chromatography, and carried out by quadrupole-orbitrap mass spectrometer (LC-MS). The detection of the characteristic amino acid sequence of BADE, the measured amino acid sequence is as follows:

[0063] 1: DWINLVYSTAHTFFYDDGK

[0064] 2: FDGIATNAVFAWVGER

[0065] The characteristic amino acid sequence of BADE in the present invention is not limited to the above sequence, and it also includes other short-chain amino acid sequence fragments, as long as the fragments are obtained by detecting BADE by LC-MS or other methods.

Embodiment 3

[0066] Cloning of embodiment 3 aflatoxin degrading enzyme BADE coding gene

[0067] 1. Primer Design

[0068] According to the characteristic amino acid sequence of BADE obtained in Example 2, it was compared in the Uniprot database. Select the corresponding protein registered in the Uniprot database, query its nucleic acid sequence, and compare it with the nucleic acid translation product according to the mass spectrometry information, and use Primer5.0 software to design and screen a pair of PCR primers for the full-length sequence of the target gene:

[0069] Upstream primer F: 5′-ATGAAACTGGCCTTAGATC-3′

[0070] Downstream primer R: 5′-TTATGCCATGCCTAATTC-3′

[0071] 2. Extraction of Bacillus subtilis Genomic DNA

[0072] Inoculate Bacillus subtilis ANSB060 in milk powder preservation into liquid LB medium for activation twice, take 3 mL of the activated bacterial liquid and centrifuge at 13 000 r / min for 1 min, discard the supernatant, add 500 μL of cell lysate, vortex t...

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Abstract

The present invention provides aflatoxin degrading enzyme secreted by Bacillus subtilis, and applications thereof. According to the present invention, the aflatoxin degrading enzyme BADE is firstly separated and purified from Bacillus subtilis ANSB060, wherein the amino acid sequence is represented by SEQ ID NO:1, and the gene encoding the enzyme is obtained through cloning; the gene is transformed into an Escherichia coli expression system so as to successfully achieve the expression of the protein in Escherichia coli; and the activity identification results prove that the recombinant rBADE has aflatoxin degrading effect, such that the foundation is established for the development of the aflatoxin degrading enzyme with high specificity and high transformation efficiency.

Description

technical field [0001] The invention relates to the technical fields of microbiology and genetic engineering, in particular to an aflatoxin degrading enzyme secreted by Bacillus subtilis and its application. Background technique [0002] Aflatoxins (AF) are toxic secondary metabolites mainly produced by fungi of the genus Aspergillus, such as Aspergillus flavus and A. parasiticus, which are highly toxic, carcinogenic and mutagenic , a serious threat to animal production and human health. The target organ of AF is the liver, which can inhibit liver lipid metabolism and protein synthesis, cause liver damage, and lead to decreased liver function. The main symptoms of liver aflatoxin poisoning include liver necrosis, hemorrhage, bile duct hyperplasia and diffuse liver fibrosis. AF can also lead to the decline of animal production performance, cause oxidative damage to animal bodies, and reduce animal immunity, making it easy to be infected by infectious pathogens. Long-term ex...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/70C12N1/21A23L5/20C12R1/19
CPCC12N9/00
Inventor 计成马秋刚赵丽红贾如
Owner CHINA AGRI UNIV
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