| Summary: | <i>Bacillus subtilis</i> SH21 was observed to produce an antifungal protein that inhibited the growth of <i>F. solani</i>. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC–MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of <i>F. solani</i> by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against <i>F. solani</i> was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of <i>Streptomyces sp.</i> N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.
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