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A kind of β-glucanase and its application in inhibiting Ochrax aspergillus

A technology of glucanase and ochrax, which is applied in the biological field to achieve low cost, avoid separation and purification steps, and reduce the risk of mildew and toxin contamination

Active Publication Date: 2022-04-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research report on the inhibition of ochrax growth by β-glucanase

Method used

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  • A kind of β-glucanase and its application in inhibiting Ochrax aspergillus
  • A kind of β-glucanase and its application in inhibiting Ochrax aspergillus
  • A kind of β-glucanase and its application in inhibiting Ochrax aspergillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Inhibition of B.subtilis CW14 thallus and its metabolites on the growth of ochrax

[0056] Use confrontation culture to study the inhibition of ochrax by the bacteria, inoculate A.ochraceus AS 3.4412 bacteria block with a diameter of 5 mm on the center of the PDA plate, and inoculate B. subtilis CW14 bacteria at a distance of 3 cm. After culturing for 5 days, measure the The farthest distance (r) from the center of the Aspergillus block to the edge of the mold colony and the distance (r') from the center of the mold to the edge of the end facing the bacteria, the inhibition rate (%) is calculated according to the following formula: Inhibition rate (%) =[(r-r') / r]×100.

[0057] The result is as figure 1 As shown, by measurement and calculation, the bacteriostatic rate of B. subtilis CW14 thallus to the growth of Ochraus persica reached 85.7%.

[0058] Streak the frozen B.subtilis CW14 strain on the LB plate, and culture it in a bacterial incubator at 37°C and...

Embodiment 2

[0062] Example 2 Screening of antifungal proteins in B.subtilis CW14

[0063] According to the detection result obtained in Example 1, the antibacterial effects of the three components with molecular weight10kDa are compared, the results show that the antibacterial effect of the component>10kDa is the best, and contains large Molecular antifungal proteins, therefore fractions >10kDa were subjected to mass spectrometry. According to the genome-wide data of B. subtilis CW14, the β-glucanase (β-GLU) of B. subtilis CW14 may have a higher activity of inhibiting the growth of Aspergillus ochrax through bioinformatics analysis combined with the results of mass spectrometry.

[0064] Analyze the signal peptide and mature amino acid sequences of the protein according to the signal peptide software (signal P-4.1Server), the full-length amino acid sequence of β-glucanase is shown in SEQ ID NO.1, and the mature peptide sequence is shown in SEQ ID NO .2, the coding gene sequence is shown ...

Embodiment 3

[0065] Example 3 Heterologous expression of β-glucanase of B. subtilis CW14

[0066] The β-glucanase of B. subtilis CW14 was heterologously expressed in Pichia pastoris, and the specific method was as follows:

[0067] (1) Construction of the recombinant expression plasmid of β-glucanase

[0068] According to the codon preference of Pichia pastoris (P. pastoris) and combined with artificial codon optimization, the codon optimization of the β-glucanase coding gene of B. subtilis CW14 was carried out, and the HIS tag and the double tag were added to the optimized sequence. Restriction sites EcoR I and Not I were obtained to obtain the sequence shown in SEQ ID NO.4. Using the pPIC9K plasmid as a vector, insert the sequence shown in SEQ ID NO.4 into the pPIC9K plasmid to construct the β-glucanase recombinant expression plasmid pPI9K-β-glu. The recombinant expression plasmid map is as follows image 3 shown. The construction of the recombinant plasmid was completed by Beijing Ao...

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Abstract

The invention relates to the field of biotechnology, in particular to a β-glucanase and its application in inhibiting Aspergillus ochrax. The present invention finds that the β-glucanase has higher activity of antagonizing Ochracus by screening components of the metabolites of Bacillus subtilis CW14 that exert antagonism against Ochraus japonicus. By adding β-glucanase to the sample to be treated, the production and growth of Ochraus can be significantly inhibited, greatly reducing the risk of mildew and toxin contamination. The invention also realizes efficient heterologous expression of recombinant β-glucanase by optimizing the expression system, and provides an effective method for obtaining a large amount of recombinant β-glucanase. β-glucanase can be used in practice to prevent and control the pollution of agricultural products by Ochratoxin and its toxins.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a beta-glucanase and its application in inhibiting Aspergillus ochrax. Background technique [0002] As one of the important fungi that cause mildew in agricultural products, Aspergillus ochraceus can cause mildew in the growth, harvest, transportation, storage and processing of grain and oil crops, and cause unavoidable economic losses; the secondary The metabolite Ochratoxin (Ochratoxin A, OTA) is highly toxic, widely polluted, and seriously harmful to human and animal health. The microbial method has become a promising strategy for reducing mold pollution because of its high efficiency, low toxicity, strong specificity, and environmental friendliness. Therefore, the development and use of antagonistic bacteria or their metabolites to inhibit the growth of toxin-producing fungi to control The method of mildew and toxin pollution is of great significance. [0003] The fibrous core...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N9/42C12N15/56C12N15/81A23L3/3571
CPCA23L3/3571C12N9/244C12N15/815C12Y302/01006A23V2002/00A23V2200/10
Inventor 梁志宏赵萌
Owner CHINA AGRI UNIV
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