Kinetic properties of β-N-acetylhexosaminidase from tobacco plants

β-N-acetylhexosaminidase from tobacco leaves (Nicotiana tabacum L.) was partially purified to final specific activity 1,72 µmol . min-1 . mg-1 using p-nitrofenyl-β-N- acetyl-D-glucosaminide as substrate. The enzyme exhibited one band after both isoelectric focusing and native electrophoresis. Molecular mass of native enzyme was determined by gel chromatography (MR 275000) and native electrophoresis (MR 285000). Isoelectric point pI 5.4 was determined by isoelectric focusing. Activity of β-N-acetylhexosaminidase was measured using substrates p-nitrofenyl-β-N-acetyl-D-galactosaminide, p-nitrofenyl-β- N-acetyl-D-glucosaminide, N,N'-diacetylchitobiose, p-nitrofenyl-N,N'- diacetylchitobioside and N,N',N''-triacetylchitotriose. For substrates N,N'- diacetylchitobiose, p-nitrofenyl-N,N'-diacetylchitobioside and N,N',N''-triacetylchitotriose an enzyme assay of β-N-acetylhexosaminidase using capillary zone electrophoresis was developed. Optimal pH and temperature of β-N-acetylhexosaminidase were determined with individual substrates, as well as products of hydrolysis. Activity of β-N- acetylhexosaminidase was highest using p-nitrofenyl-β-N-acetyl-D-glucosaminide as substrate and lowest using N,N',N''-triacetylchitotriose (35% in relative comparison). Maximum velocity and Michaelis constant of..