Purification and properties of N-acetyl--D glucosaminidase from bovine testicle

N-Acetyl-β-D-glucosaminidase (NAGase) from bovine testicle was purified by ammonium sulfate fractionation followed by diethylaminoethyl (DEAE)-cellulose (DEAE-32) and Sephacryl S-300 chromatography. The enzyme was purified to homogeneity as analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing gel electrophoresis (IFGE). The specific activity of the purified enzyme was 658.21 U/mg. The enzyme was a single subunit with molecular weight of 68.3 kDa and contained 3.03% sugar. The pI value was calculated to be 5.54 using IFGE. The optimal pH and temperature of the enzyme for hydrolysis of p-Nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be pH 5.6 and 50°C, respectively. The kinetics results showed that the enzyme hydrolyzed pNPNAG following Michaelis-Menten with Km of 0.71 mM and Vm of 16.72 M/min at pH 5.6 and 37°C. The enzyme was stable at pH values ranging from 2 to 6.5 and at temperatures below 60°C. The activation energy was determined to be 64.19 kJ/mol.    Key words: Bovine testicle, N-Acetyl-β-D-glucosaminidase, purification, characteristic.