Isolation, Purification And Characterization Of N-Acetyl-β-D-glucosaminidase From Bovin Testes

N-Acetyl-β-D-glucosaminidase (NAGase,EC3.2.1.52) was purified from Bovin testes by extraction with 0.01M Tris-HCl buffer (pH 7.5) containing 0.2M NaCl, ammonium sulfate fractionation, and then by chromatography on DEAE-cellulose (DEAE-32) and Sephacry S-300.The purified enzyme showed a single band on polyacrylamide gel electrophoresis,and the specific activity was determined to be 658.21 U·mg¹.The pI value was revealed to be 5.54 by isoelectric focusing. The molecular weight of the whole enzyme was found to be 68.3 kDa ,it is consisted of 364 amino acid residues.The enzyme was found to be made up of only 1 subunit and detected to host several disulphide bonds. The NAGase was a glycoprotein with 3.03%(W/W)glucose. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl- N-acetyl-β-D-glucosaminide (pNP-NAG)were found to be at pH 5.6 and at 50°C, respectively. The enzyme is stable within the pH range of 3.0 to 6.6 and at temperatures below 55°C.The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis–Menten kinetics with Km of 0.71 mmol·L^-1 and Vm of 16.72μmol·L^-1·min^-1at pH 5.6 and 37°C. The activation energy was determined to be 64.19 kJ·mol^-1.The effects of metal ions on the enzyme were studied. The results showed that Na⁺,Cu²⁺ can inhibit enzyme activity slightly. Mg²⁺,Ca²⁺,Zn²⁺,Mn²⁺ were reversible competitive inhibitors which reduce enzyme activity to different degrees. The inhibition constants are 75.26mmol·L^-1, 87.37 mmol·L^-1, 2.21 mmol·L^-1and 41.18 mmol·L^-1, respectively.The effects of 15 kinds of amino acids on the enzyme were studied. The results showed that L-Ile,L-Met,L-Phe,L-Ala,L-Leu,L-Pro,L-Thr,L-Gly,L-Gln,L-Ser,L-Asn and L-His had not any effect on enzyme activity in the tested range of concentration. L-Val can increase the activity of enzyme slightly. L-Arg and L-Lys reduced the activity of enzyme at different concentration. L-Arg was revealed to be a reversible non-competitive inhibitor with a KI of 24.22mmol·L^-1. L-Lys was reversible anticompetitive inhibitor . The KI was 3.597 mmol·L^-1.The effects of different components of semen diluter were studied. The results showed that, Benzylpenicillin sodium and Streptomycin sulfate had no effect on enzyme activity. Sodium citrate can reduce the enzyme activity slightly. Potassium chloride activated the enzyme at low concentrations, but inactivated it at high concentration.Glucose and baking soda inhibited enzyme activity greatly, while glucose was found to be reversible competitive inhibitor(KI = 0.36 mol·L^-1) and Baking soda was a reversible anticompetitive inhibitior (KI =7.01 mmol·L^-1) .