Studies On Regulation Of The N-Acetyl-β-D-glucosaminidase From Helicoverpa Armigera

Aβ-N-Acetyl-D-glucosaminidase (EC3.2.1.52) was purified from the pupae of Helicoverpa armigera by using ammonium sulfate precipitation, and column chromatography on Sephadex G-200 and DEAE-Cellulose. The purified enzyme was migrated as a single band on native polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 89.3 kDa. The optimum pH was determined to be at pH 5.6 and the optimum temperature was at 55℃. The enzyme was stable in the range pH4.0-8.0, and inactivated above pH8.0 quickly under 37℃. The enzyme was heat-stable under 45℃after 30min.The effects of several metal ions, organic solvents and saccharide substances on the enzyme activity had been studied. The results indicated that the methanol, ethanol, propanol, glycol and glycerol activated the enzyme at low concentrations, but they inactivated it at high concentrations. The results showed that the inactivation of the enzyme by these substances was reversible reaction, except Hg²⁺. Cu²⁺, glucose and galactose were mixed type inhibitors. Zn²⁺,Al³⁺, acetone, dimethyl sulfoxide , dioxane ,DMF and fructose were competitive type inhibitors. Pb²⁺ and sucrose were a noncompetitive type inhibitors. NAG was a reverse competitive type inhibitor.The characters of functional groups of the enzyme active site had been studied using the kinetic method and chemical modification. The result showed that the residues of tryptophan, histidine, acidic amino acids and lysine were essential for the enzyme catalytic activity. The pKe value of the ionization of the enzyme active site was determined to be 4.59, and theΔH⁰ value to be 1.04 kcal/mol. The result suggested that the ionization group be the carboxyl-group of acidic amino acids.The conformational changes of the enzyme in presence of guanidine hydrochloride at different concentrations were studied by means of measuring the fluorescence emission spectra. The fluorescence intensity of the enzyme gradually decreased with increasing guanidine concentrations. The fluorescence emission peak(at 341 nm) of the enzyme in 5 mol/L guanidine solution was red-shifted to 351 nm. It is suggested that the enzyme molecule has spread sufficiently, and the enzyme has inactivated totally. Inactivation and unfolding of the Helicoverpa armigera...