Biochemical analyses of a new GH18 chitinase from Beauveria bassiana KW1 and its synergy with a commercial protease on silkworm exuviae hydrolysis

ABSTRACT This study aimed to investigate the biochemical characteristics of a new GH18 chitinase (BbChi3250) derived from Beauveria bassiana KW1, which was isolated from Bombyx batryticatus. BbChi3250 occurs widely throughout Beauveria and Cordyceps species and has low sequence identity (≤39.58%) with already-characterized B. bassiana chitinases. Biochemical analyses revealed that BbChi3250 is an endo-chitinase and shows good activity toward ethylene glycol chitin, with a kcat/Km of 596.00 mL·mg–1·s–1. Importantly, BbChi3250 demonstrates significant synergy with protease CbPro in hydrolyzing silkworm exuviae, reflected in high degrees of synergy in releasing N-acetyl-β-D-glucosamine and tyrosine equivalents. Scanning electron microscopy analysis of enzyme-digested silkworm exuviae confirmed the enzymes' surface ablation activities and mutual synergy. To the best of our knowledge, this is the first report about the synergistic degradation of silkworm exuviae in vitro by protease and B. bassiana-sourced chitinase, providing new insights into the enzyme’s properties and applications in the degradation of insect cuticles.IMPORTANCEB. bassiana, an entomopathogenic fungus, is utilized in producing Bombyx batryticatus, a traditional Chinese medicine for treating stroke and related symptoms. This is achieved by infecting Bombyx mori larvae with B. bassiana. Chitin, a key component of the silkworm cuticle, is hydrolyzed by endo-chitinase, a critical virulence factor of B. bassiana. Despite the presence of multiple chitinase-encoding genes in B. bassiana strains, such as the twenty GH18 genes in ARSEF 2860, only a few have been studied. Further research on these chitinases could elucidate B. bassiana’s pathogenic mechanisms and uncover chitinases with new properties.