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Abstract
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 ïM/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.  
Highlights
N-Acetyl-β-D-glucosaminidase (NAGase, EC 3.2.1.52), an intracellular lysosomal hydrolase, is widely distributed in nature (Liu et al, 2009; Keyhani et al, 1996)
The enzyme was purified to homogeneity as analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing gel electrophoresis (IFGE)
We found that the purified NAGase is bound to the concanavalin A (Con A) column
Summary
N-Acetyl-β-D-glucosaminidase (NAGase, EC 3.2.1.52), an intracellular lysosomal hydrolase, is widely distributed in nature (Liu et al, 2009; Keyhani et al, 1996). An in-depth study of NAGase can provide a basis for life science research. Research reports have shown that NAGase has been investigated more in microbial systems and has been the focus of a large number of gene cloning and expression studies (Mamarabadi et al, 2009). Using muscovy duck testicles as a source, NAGase was extracted, and some of its enzymatic properties and the effect of metal ions on enzyme activity have been determined (Wang et al, 2007). High NAGase activity is present in rabbit semen (Farooqui and Srivastava, 1980), human sperm (Perez et al, 2007), and ascidian eggs (Koyanagi and Honegger, 2003). We purified NAGase from bovine testicle and studied the enzymatic characteristics to provide further details on the physiological functions of NAGase in bovines
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