Protease and beta-N-acetylglucosaminidase of honey bee chalkbrood pathogen Ascosphaera apis

Lytic enzymes are usually reported to have a role in fungal entomopathogenicity. Enzymic profiles produced by Ascosphaera apis, a pathogen causing chalkbrood disease in honey bee larvae were determined by API ZYM kit and standard enzyme assays. Ten isolates of A. apis produced 11 enzymes (i.e. protease, beta-N-acetylglucosaminidase, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-B1-phosphohydrolase, beta-glucosiclase and alpha-mannosidase). Two main enzymes (protease and beta-N-acetylglucosaminidase), that might play roles in either penetration of the peritrophic membrane in bee larval midgut or breaking down the cuticle of larvae were chosen for further study. All isolates of A. apis gave the highest proteolytic enzymes in sterile germination medium after 11 day-incubation at 30 degrees C. Yeast extract and glucose were the key components for the fungal growth and protease production. The pH optimum for protease production was 8.0. Phenylmethanesulphonyl fluoride (PMSF) and 1, 10-phenanthroline could markedly inhibit protease activities, indicating serine- and metal loproteases. In our studies on beta-N-acetylglucosaminidase, we found that A. apis HL-5-2 grown in enrichment culture medium containing 0.2% colloidal chitin at 30 degrees C for 14 days, produced the highest yield of enzyme. Purification of beta-N-acetylglucosaminidase was carried out using ammonium sulphate precipitation, ion exchange (DEAE-sepharose) and gel filtration (Sephacryl S-200 HR) column chromatography. After ion-exchange chromatography, beta-N-acetylglucosaminidase had a specific activity and recovery yield of 42.8 and 8.4 % respectively. The partially purified enzyme was a monomer with a molecular weight of 55 kDa, with pH and temperature optima of about 5.5 and 35 degrees C. Several cationic compounds (e.g. Ca2+, Mg2+, K+, Na+ and Li+) slightly reduced beta-N-acetylglucosaminidase activity whilst Cu2+ and Zn2+ significantly decreased its activity to 50.31 and 91.08 % respectively. The enzyme was stable at 30-37 degrees C and pH 5.0-7.0.