Molecular Identification and Recombinant Expression of a Novel Antifungal Protein from Wheat-Associated Paenibacillus
Xiaohong Ge1, Zhikun Chen1, Haoyuan Guo2
1School of Food Science, Henan Institute of Science and Technology, Xinxiang Engineering Technology Research Center for Agricultural Products Processing, Research and Experimental Base for Traditional Specialty Meat Processing Techniques of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, Xinxiang 453003, China.
Abstract:
Fusarium head blight (FHB) caused by Fusarium graminearum leads to huge yield losses and mycotoxin contamination in wheat globally. Paenibacillus polymyxa with strong antagonistic activity was preliminarily identified. To clarify the key antifungal component, an extracellular protein was purified via ammonium sulfate precipitation, DEAE-52 anion-exchange and Sephadex G-75 gel filtration chromatography. SDS-PAGE showed a single band at 76 kDa. liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed this protein belongs to glycosyl hydrolase family with 86% sequence coverage. Biochemical characterization showed that the crude protein was stable at 40-90 °C and pH 3.0-9.0, sensitive to proteinase K, trypsin and neutral protease. The purified 76 kDa protein exhibited antifungal activity against F. graminearum. The gene encoding this protein was cloned and expressed in Escherichia coli. The renatured recombinant protein p76kd showed comparable antifungal activity to the native protein. This study purified and characterized a 76 kDa protein annotated as a glycosyl hydrolase via LC-MS/MS peptide matching; its antifungal function is presumed to originate from the conserved glycosyl hydrolase domain according to existing homologous research, which is distinct from previously reported lipopeptides or uncharacterized complexes. This protein provides a promising candidate for the biocontrol of FHB and related fungal diseases in cereal crops.
