Study on Purification and Properties of the Alkaline N-Acetyl-β-D-glucosaminidase from Porcine Semen

【Objective】The purpose of this experiment was to separate and purify the N-Acetyl-β-D-Glucosaminidase (EC3.2.1.30,NAGase) from the porcine semen. The physicochemical properties of the enzyme were studied. 【Method】 The NAGase was purified by ammonium sulfate fractionation, chromatography on DEAE-32, Sephadex G-100 and CM-52. The purity of the enzyme was identified by polyacrylamide gel electrophoresis. The pI value was determined by isoelectric focusing. The molecular weight of the subunit of the enzyme was determined by SDS-polyacrylamide gel electrophoresis.【Result】The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis. The specific activity of the enzyme was of 17 606.15 U·mg-1. The molecular weight of the subunit of the enzyme was determined to be 58.03 kD. The pI value was calculated to be 9.42. The optimum temperature and pH of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP- NAG) were determined to be at 50℃ and at pH 5.5, respectively. The enzyme was stable in the pH ranges of 3.0 to 8.9 under 37℃ and at temperatures below 45℃. The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of pNP-NAG. The Km and Vm values were determined to be 1.94 mmol·L-1 and 27.53 μmol·L-1·min-1 at pH 5.6 and 37℃, respectively. The activation energy of the enzyme for the hydrolysis of pNP-NAG was to be 88.73 kJ·mol-1. 【Conclusion】The method used to separate and purify the enzyme in this experiment was feasible.