The Properties And Activity Regulation Of N-Acetyl-β-D-glucosaminidase From The Sperm Of Huai-pig

N-Acetyl-β-D-glucosaminidase （EC3.2.1.52） was purified from the sperm of Huai-pig.The purification steps involved the following procedures: ammonium sulfate precipitation,anion-exchange chromatography on DEAE-32, gel filtration chromatography on Sephadex G-200and cation-exchange chromatography on CM Sepharose Fast Flow. The purified enzyme showed asingle band on polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Thespecific activity was2759.15U mg^-1and recovery was7.88%. The molecular weight wasestimated to be71kDa, only consisting of one subunits according to gel filtration chromatographyand SDS-PAGE, and the isoelectric point was calculated to be8.9.The optimum pH was5.7, the optimum temperature was at50℃for the hydrolysis ofp-nitrophenyl-N-acetyl-D-glucosaminide （pNP-GlcNAc） by the enzyme. However, the optimumpH and optimum temperature of the enzyme for the hydrolysis of pNP-GalNAc were found at pH5.6and at55℃, repectively. The kinetic behavior of the enzyme in the hydrolysis of pNP-GlcNAcand pNP-GalNAc followed Michaelis-Menten kinetics with Kmof0.80mmol L^-1and0.24mmol L^-1, Vmof25.16μmol L^-1 min^-1and3.68μmol L^-1 min^-1, repectively. The enzyme activitywas unaffected after exposure to temperatures below50℃, as well as to pH3.0-8.6. The activationenergy was69.09kJ mol^-1for the hydrolysis of pNP-GlcNAc by the enzyme.The characters of functional groups of the enzyme active site were studied using chemicalmodification and the kinetic method. The pKevalue of the enzyme active site was determined tobe5.29at37℃, and the ΔHovalue to be7.28kcal mol^-1. The result showed that the ionzationgroup was imidazole-group of histidine. The enzyme was modified under special conditions, theresult suggested that disulfide bond, sulfydryl-group of cysteine, indolyl-group of tryptophan andamino-group of lysine were also essential for the catalytic activity of the enzyme, but the residueof arginine was not necessary.The inhibitions of the enzyme by the products were studied using the kinetic mothod, whichshowed that the first product NAG was a mixed type inhibitor, while the second product phenolof pNP analogues had no influence on the enzyme activity. The catalyze mechanism of theenzyme for the hydrolysis of pNP-GlcNAc belonged to ordered Bi Bi according to Cleland rule. The effects on the enzyme activity by some components in seminal plasma were studied. Theresults showed that Na⁺、K⁺、Cl^-and lower concentration fructose had no effect on the enzymeactivity. HCO-3、PO3-4、alkaline amino acid、vitamin B1and higher concentration fructose showedvarious degrees of inhibitory effects on the enzyme activity. Citric acid activated the enzyme atfirst, then inhibited it, and vitamin C was significantly increase the enzyme activity. The catalyzemechanism were studied using the kinetic mothod, the results suggested that Lys、Arg、His andHCO-3were competitive type inhibitors, the KIwere16.92mmol L^-1、28.13mmol L^-1、77.43mmol L^-1and48.54mmol L^-1, repectively; fructose was a mixed type inhibitor, the KIand KISwere0.89mol L^-1and0.43mol L^-1, repectively; PO43-and citric acid were non-competitive typeinhibitors, the KIwere31.48mmol L^-1and63.58mmol L^-1, repectively; vitamin B1was aun-competitive type inhibitor, the KISwas1.69mmol L^-1; and vitamin C was a partlycompetitive type activator.The effects on the enzyme activity by some microelements were studied. The results showedthat Mn2+、Co2+and I-activated the enzyme under lower concentrations. Fe²⁺、Zn²⁺、Cu²⁺、Cr³⁺and higher concentration Mn2+showed significantly inhibitory effects on the enzyme activity, andthe degree of inhibition was Cr³⁺> Cu²⁺> Fe²⁺> Zn²⁺> Mn²⁺. However, Sodium selenite activatedthe enzyme at first, then inhibited it. Their inhibition types and inhibition constants weredetermined. The results revealed that Cr³⁺、Cu²⁺、Fe²⁺、Zn²⁺and Mn²⁺ were non-competitive typeinhibitors, and the KISwere7.68μmol L^-1、0.11mmol L^-1、1.14mmol L^-1、3.15mmol L^-1and59.18mmol L^-1, repectively. Sodium selenite was a mixed type inhibitor, the KIand KISwere0.15mmol L^-1and0.53mmol L^-1, repectively. The results also showed that the enzyme thermalstability was reduced in certain concentrations of Cu2+and Zn2+, and EDTA2Na activated theenzyme activity.