Isolation,Purification And Characterization Of β-N-Acetyl-D-glucosaminidase From Helicoverpa Armigera

A β-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 and SDS-polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 89. 3 kDa. The Specific activity of the enzyme was 2678.79 U/mg. The kinetic behavior of this enzyme was performed using pNP-β-D-GlcNAc as substrate. The optimum pH was determined to be at pH 5.63 and the optimum temperature was at 55 ℃. The enzyme was stable in the range 4.0-8.0, and inactivated above 8.0 quickly under 37℃. The enzyme was heat-stable under 50℃ after 30min. The enzyme follows typical Michaelis-Menten kinetics for the hydrolysis of pNP-P-D-GlcNAc and the K_m and V_m values were determined to be 0.16 mmol/L and 10.73 μmol/L/min, respectively. The activation energy with pNP-P-D-GlcNAcwas 66.24 KJ/mol.Another β-N-Acetyl-D- glucosaminidase (EC3.2.1.30) were isolated from the larva and adult of Helicoverpa armigera (Hubner), and partially purified by ammonium sulfate fractionation and dialysis. The specific activity of the enzyme was 37.33 and 47.69 U/mg, 3.64 and 2.35 fold purification were achieved, respectively. Michaelis-Menten kinetics for P-N-Acetyl-D- glucosaminidase from the larva and adult, the K_m values were 0.126 and 0.2 mmol/L , the V_m values were 2.51 and 10.18 μmol/L'min, respectively.The effects of metal ions on β-N-acetyl-D-glucosaminidase (NAGase, EC3.2.1.52) from Helicoverpa armigera were studied. The results showed that Na+, K+ and Li+ ions had no any effects on the enzyme activity. Mg²⁺, Ca²⁺ and Co²⁺ ions had few effects on the enzyme activity, while Zn²⁺, Cu²⁺, Cd²⁺, Al³⁺, Hg²⁺ and Pb²⁺ inhibited the enzyme. The inhibitory kinetics of Cu²⁺, Al³⁺, Zn²⁺, Hg²⁺ andPb2+ on the enzyme were further studied. The results showed that Cu2+ was a reversible competitive inhibitor of the enzyme and was determined to be a mixed type inhibitor. The inhibition constants of free enzyme (K\) and enzyme-substrate complex (Kis) with Cu2+ were determined to be 0.23 mmol/L and 1.49 mmol/L, respectively. Al + was a reversible competitive inhibitor of the enzyme, and the inhibition constants (K\) was determined to be 6.42 mmol/L. The inhibition of Hg + on the enzyme underwent a reversible reaction, and was a mixed-type inhibitor. The inhibition constants of free enzyme (Ki) and enzyme-substrate complex (Kis) with Hg2+ were determined to be 0.23 mmol/L and 1.49 mmol/L, respectively. Zn2+ was reversible competitive inhibitor. The inhibition constants of free enzyme (K\) with Zn2+ was 1.58 mmol/L. The enzyme was heat-stable in the range 30-40 °C in the present of Zn2+.After treatment with metal chelating agent-ethylenediaminetetraacetic acid disodium (EDTA), the enzyme activity was completely lost, which indicated that the activity of P-N-Acetyl-D-glucosaminidase was correlated with some metalions.Effects of different organic solvents on the enzyme activity and conformation of P-N-Acetyl-D-glucosaminidase were investigated. The results indicated that the methanol, alcohol and glycol activated the enzyme at low concentrations, but they inactivated it at high concentrations. Propanol and glycerol inactivated the enzyme. Formaldehyde can inhibited the activity of enzyme, and it was a mixed-type inhibitor. The inhibition constants of free enzyme (K{) and enzyme-substrate complex (K\s) with Hg2+ were determined to be 0.51 mmol/L and 0.24 mmol/L, respectively. Acetone can induced the enzyme to reversible inactivate in the present of the low concentration of acetone. The values of Vmax decreased gradually along with the increase of acetone concentration, however, the value of Km kept unchanged . At the same time, the conformational changes of the enzyme in acetone solutions of different concentrations weremeasured by fluorescence absorption spectra. The fluorescence emission peak in tensity of the enzyme gradually weakened with increasing acetone concentrations, accompanied by the peak being gradually blue-shifted. The result showed that acetone could effect the conformation of the enzyme. The emission peak of the native enzyme was at 339nm, and it decreased in intensity and blue shifted with the increasing concentrations of Cu2+. This showed that Cu2+ changed the conformation of enzyme. Dimethyl sulfoxide and dioxane inhibited p-N-Acetyl-D-glucosaminidase. Dimethyl sulfoxide was a reversible competitive inhibitor, and the inhibition constants of free enzyme (K{) was 3.46 mmol/L. Dioxane was a reversible competitive inhibitor, too. They both changed fluorescence emission spectra and UV absorption spectra. The emission peak at 339nm decreased in intensity with the increasing concentrations of dimethyl sulfoxide. When the concentration of dimethyl sulfoxide above 30%, the intensity of UV absorption increased.The emission peak at 339nm decreased in intensity with the increasing concentrations of guanidine solution. When the concentration of guanidine solution was 2 mol/L, The fluorescence emission peak at of the enzyme was red-shifted to 355 nm.When Try residue of the enzyme was modified by NBS, the connection between Try residue and activity of enzyme could be studied according to the fluorescence emission spectra and the ultraviolet spectra. The result showed that The fluorescence emission peak at 339nm and the ultraviolet absorbed peak at 278nm were disappeared with the loss of the activity of the enzyme after the enzyme was modified by NBS.The thermal stabilities of the enzyme was studied. The results showed that the enzyme was heat-stable below 50°C and starts thermal denaturation at 55 °C. The activity of the enzyme lost 50 percent by the treatment at 67°C for 30min. And, the enzyme was totally inactivated by the treatment above 80 °C. The kinetics of thermal inactivation of the enzyme at high temperature was further studied. The...