Partial purification and characterization of a β- N-acetylhexosaminidase from black cherry ( prunus serotina EHRH.) seeds

Abstract

A β- N-acetylhexosaminidase (β-NAHA) was purified 30-fold from hemogenates of mature black cherry seeds by DEAE-cellulose and Concanavalin A-Sepharose 4B chromatography. Extensive further purification was achieved by N- acetyl- D-glucosamine-agarose chromatography, but the enzyme thereafter required bovine serum albumin (BSA) for retention of activity. The purified enzyme showed highest acivity p- nitrophenyl-N- acetyl-β- D- glucosaminide (PNP-βGlcNAc) (optimum pH 4.5; K m, 0.49 mM) and p-nitrophenyl- N-acetyle- β-D-galactosaminide (PNP-β-GalNAC) (optimum pH 4.0; K m , 0.63 mM). Mixed-substrate experiments suggested that these substrates compete for the same active site. Lesser activity was shown towards p-nitrophenyl- and o- nitrophenyl-β- D- glucosides . p- Nitrophenyl-N- acetyl-α- D- glucosaminide , p- nitrophenyl-N- acetyl-α- D- mannosaminide and p- nitrophenyl-α- D- glucoside were not hydrolysed. The enzyme preparation lacked amygdalin hydrolase, prunasin hydrolase and p nitrophenyl-α- D- mannosidase activities and failed to release N-acetylglucosamine from a glyco-protein fraction derived from black cherry seeds by Concanavalin A-Sepharose 4B chromatography. β-NAHA activity was not stimulated by any metal ions tested and was unaffected by diethyldithiocarbamate, 2,2′-dipyridyl, 1,10-phenanthroline, iodoacetate and iodoacetamide.