Isolation, Purification And Properties Of Isoenzyme Of N-Acetyl-β-D-glucosaminidase From Sperm Of Pork Type

Alkaline and Acidic N-Acetyl-β-D-glucosaminidase(NAGase,EC3.2.1.52)were purified from pig semen,by extraction with ammonium sulfate fraction,and then chromatography on DEAE-32,Sephades G-100 and CM-52.The two purified enzymes were a single band on polyacrylamide gel electrophoresis with specific activity to be 17606.15 U/mg and 1773.25 U/mg,respectively.The Alkaline enzyme had only one kind subunit,molecular weight of which was determined to be 64.30 kD;Acidic enzyme had two kind subunits and molecular weight were 129.13 kD and 62.24 kD.The pI value were determined to be 9.42 and 5.10 by isoelectric focusing,respectively.The optimum pH of the two enzymes for the hydrolysis of p-Nitrophenyl-N-Acetyl-β-D-glucosaminide (pNP-NAG)were determined to be at 5.50,the optimum temperature were determined to be at 50℃and 60℃,respectively.The result of the stability showed that alkaline enzyme was stable at the pH range from 3.0 to 8.90 and at the temperature below 45℃,and acidic enzyme was stable at the pH range from 3.6 to 9.20 and at the temperature below 55℃The kinetic behavior of the alkaline enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with K_m of 1.94 mmol/L and V_m of 27.53 umol/L/min at pH 5.63 and 37℃.The activation energy was determined to be 88.73 KJ/mol.For the acidic enzyme,when[S]＜0.7 mmol/L,followed Michaelis-Menten kinetics with K_m of 0.46 mmol/L and V_m of 17.34 umol/L/min at pH 5.60 and 37℃,the activation energy was determined to be 41.70KJ/mol.Substrate,when[S]＞0.7 mmol/L, can inhibit enzyme activity.The effects of metal ions on the alkaline NAGase were studied.The results showed that Cu²⁺,Zn²⁺,Mg²⁺,Ca²⁺and Na⁺,K⁺ at high concentration(0.1～1mol/L) showed various degrees of inhibitory effects on the enzyme.Na⁺,K⁺,Zn²⁺and Ca²⁺were reversible non-competitive inhibitor.The inhibition constants of free enzyme(K₁)with Na⁺ and K⁺ were 0.47 mol/L and 0.32 mol/L,respectively.The inhibition constants of free enzyme(K₁)with Zn²⁺was 3.27 mmol/L.The inhibition constants of free enzyme(K₁) with Ca²⁺was 65.28 mmol/L.Na⁺ and K⁺ at low concentration(1～10 mmol/L)had no any effects on the activity of enzyme.Fructose,D-Mannose and N-Acetyl-D-Glucosamine inhibited the alkaline NAGase in certain concentrations.D-Mannose was reversible competitive inhibitor.The inhibition constants of free enzyme(K₁)with D-Mannose was 18.36 mmol/L.N-Acetyl-D-Glucosamine was a mixed-type inhibitor.The inhibition constants of free enzyme(K₁)and enzyme-substrate complex(K_IS)with N-Acetyl-D-Glucosamine were determined to be 16.98 mmol/L and 55.26 mmol/L,respectively.Enrofoxacin,Clprofloxacin,Chloramphenicol,Florfenicol at low concentration (2～20μg/mL)and Sulfamete at high concentration(0.1～1 mg/mL)had no effects on the activity of alkaline NAGase.Otherwise,Enrofoxacin and Clprofloxacin can inhibite the activity of alkaline NAGase at high concentration(0.1～1.5 mg/mL).High concentration Chloramphenicol(0.2～2 mg/mL)can stick up to the inhibition of ethanol for enzyme activity.Cephradine(0.5～15 mg/mL)activated the enzyme activity at low concentration and inhibited it at high concentration.For ingredients in common usage for semen diluter,at the range of certain concentrations,EDTA2Na and VitaminC can activate the alkaline NAGase while Tris,sodium bicarbonate,glucose,trisodium citrate can inhibit the enzyme activity in some degree.Tris was the strongest inhibitor of all and was reversible non-competitive inhibitor with the inhibition constants of free enzyme(K₁)was 9.81 mmol/L. Benzylpenicillin potassium,Streptomycin sulphate and L-Cysteine had no effects on the activity of enzyme.The effects of semen collection frequency on the enzyme from seminal plasma were studied and activities of NAGase in semen from different boar species were compared.The results showed that semen collection twice a week had no effect on the enzyme activity,but the enzyme activity could be decreased because of semen collection one time every two days or one time a day.The activity of enzyme from Duroc seminal plasma were higher than Yorshire and the offsprings of Duroc and Pietrain(P＜0.05). But,there were no distinct difference on the contents of NAGase in sperm cell of those three species.