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Isolation, purification, gene cloning and expression of antifungal protein from Bacillus amyloliquefaciens MG-3
Fen Yan1, Xiaoling Ye2, Chunhua Li2
1College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian 350108, China; College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China; Key Laboratory of Postharvest Biology of Subtropical Special Agricultural Products, Fujian Province University, Fuzhou, Fujian 350002, China.
Abstract:
The antifungal protein MG-3A was isolated from Bacillus amyloliquefaciens MG-3, and was purified and identified. The results showed that antifungal protein MG-3A was likely a serine protease with a molecular weight of ~48 kDa. The serine protease exhibited a broad antifungal spectrum and effectively extended the shelf-life of loquat fruit up to 25 d. The antifungal protein MG-3A showed good stabilities to temperature, pH and protease K. Primers were designed according to the mass spectrum of antifungal protein and the comparison with proteins in the NCBI database. The serine protease gene MG-3A from B. amyloliquefaciens genome was isolated and cloned using PCR. The prokaryotic expression plasmid pET28a-MG-3A was constructed and used to express the antimicrobial protein in vitro. The SDS-PAGE results showed that the recombinant protein expressed in Escherichia coli BL21 (DE3) was highly soluble. Affinity chromatography was used to purify the recombinant protein and its antifungal activity was evaluated.
Insights
A novel antifungal protein, MG-3A, isolated from Bacillus amyloliquefaciens, demonstrates broad-spectrum activity and enhances loquat fruit shelf-life. This serine protease shows stability and was successfully expressed in E. coli.
Area of Science:
- Microbiology
- Biochemistry
- Food Science
Background:
- Antifungal proteins are crucial for controlling microbial spoilage in food.
- Bacillus amyloliquefaciens is a known source of bioactive compounds.
- Extending the shelf-life of perishable fruits like loquats is an ongoing challenge.
Purpose of the Study:
- To isolate, purify, and characterize a novel antifungal protein from Bacillus amyloliquefaciens MG-3.
- To investigate the antifungal spectrum and efficacy of the purified protein in extending fruit shelf-life.
- To clone, express, and purify the gene encoding the antifungal protein for further study.
Main Methods:
- Isolation and purification of antifungal protein MG-3A.
- Determination of protein molecular weight and identification as a serine protease.
- Antifungal spectrum analysis and loquat fruit shelf-life extension tests.
- Gene cloning using PCR and prokaryotic expression in E. coli.
- Purification of recombinant protein via affinity chromatography.
Main Results:
- Antifungal protein MG-3A, a ~48 kDa serine protease, was identified.
- The protein exhibited broad antifungal activity and extended loquat shelf-life to 25 days.
- MG-3A demonstrated stability to temperature, pH, and protease K.
- The recombinant protein expressed in E. coli was soluble and purified.
Conclusions:
- Antifungal protein MG-3A is a promising candidate for natural food preservation.
- The successful cloning and expression facilitate further research into its mechanism of action.
- This serine protease offers a potential solution to reduce post-harvest losses in fruits.
