Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain

摘要

A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad 0 Sepharose ion-exchange chromatography, and Superdex 75 FIR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N"-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degreesC, and a K-m of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degreesC. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degreesC, and a K-m of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg2+ and Ag+, p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.