GLUCOSE DEHYDROGENASE (NAD(P)-dependent) from Microorganism

PREPARATION and SPECIFICATION Appearance White amorphous powder, lyophilized. Activity GradeⅢ　250 U/mg-solid or more Contaminants NADH oxidase ≤1.0×10 -3  % α-Glucosidase ≤1.0×10 -3  % Glucose-6-phosphate dehydrogenase ≤1.0×10 -3  % PROPERTIES Stability Stable at −20 ℃ for at least one year （Fig.1） Molecular weight approx. 101,000 (Gel filtration) Isoelectric point 4.5 Michaelis constants NAD ＋ linked 1.38×10 -2  M (D-Glucose) 3.09×10 -4  M (NAD ＋ ) NADP ＋ linked 1.25×10 -2  M (D-Glucose) 4.07×10 -5  M (NADP + ) Inhibitors Ag ＋ , Hg 2＋ , Monoiodoacetate Optimum pH 9.0 （Fig.4） Optimum temperature 55 ℃ （Fig.5） pH Stability pH 6.0−7.5 (20 ℃, 16 hr) （Fig.6） Thermal stability 45 ℃ (15 min-treatment with 50 mM K-phosphate buffer, pH 7.0) （Fig.7） Substrate specificity Specific for β-D-Glucose or 2-Deoxy-glucose (Table.1) (Either NAD ＋  or NADP ＋  serves as coenzyme.) APPLICATIONS This enzyme is useful for enzymatic determination of D-glucose. ASSAY Principle The formation of NADH is measured at 340 nm by spectrophotometry. Unit definition One unit causes the formation of one micromole of NADH per minute under the conditions detailed below. Method Reagents A. Tris-HCl buffer, pH 8.0 0.1 M B. D-Glucose solution 1.5 M C. β-NAD＋ solution 80 mg/mL D. Enzyme diluent 50 mM K-phosphate buffer, pH 7.0 contg. 0.1 % BSA Procedure 1.Prepare the following reaction mixture in a cuvette (d ＝ 1.0cm) and equilibrate at 37 ℃ for approximately 5 minutes. 2.6 mL Tris-HCl buffer, pH 8.0 (A) 0.3 mL Substrate solution (B) 0.1 mL β-NAD＋ solution (C) Concentration in assay mixture Tris-HCl buffer 85.25 mM D-Glucose 147.54 mM NAD ＋ 3.66 mM 2.Add 0.05 mL of the enzyme solution* and mix by gentle inversion. 3.Record the increase in optical density at 340 nm against water for 2 to 5 minutes with a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test). At the same time, measure the blank rate (ΔOD blank) using the same method as the test except that the enzyme diluent (D) is added instead of the enzyme solution. * Dissolve the enzyme preparation in ice-cold enzyme diluent (D), dilute to 0.8−1.2 U/mL with the same buffer and store on ice. Calculation Activity can be calculated by using the following formula : Volume activity (U/mL) = ΔOD/min (ΔOD test−ΔOD blank)×Vt×df 6.22×1.0×Vs ＝ ΔOD/min×9.807×df Weight activity (U/mg) ＝ (U/mL)×1/C Vt : Total volume (3.05 mL) Vs : Sample volume (0.05 mL) 6.22 : Millimolar extinction coefficient of NADH under the assay conditions (cm 2 /micromole) 1.0 : Light path length (cm) df : Dilution factor C : Enzyme concentration in dissolution (c mg/mL) Table 1. Substrate Specificity of Glucose dehydrogenase Substrate (150mM) Relative activity(%) D-Glucose 100.0 L-Glucose 0.0 D-Xylose 16.2 2-Deoxy-glucose 127.0 L-Sorbose 0.0 D-Mannose 5.1 D-Fructose 0.0 Substrate (150mM) Relative activity(%) Galactose 1.7 D-Lactose 1.5 D-Sorbitole 0.0 D-Mannitol 0.0 Sucrose 0.0 Inositol 0.0 Maltose 1.4 Table 2. Effect of Various Chemicals on Glucose dehydrogenase ［The enzyme dissolved in 50 mM K-phosphate buffer, pH 7.0 (2.8 U/mL) was incubated with each chemical for 1 hr at 30 ℃.］ Chemical Concn.(mM) Residual activity(%) None - 100 Metal salt 2.0 AgNO 3 7.1 Ba(OAc) 2 98.2 CaCl 2 98.9 Cd(OAc) 2 96.6 CoCl 2 96.4 CuSO 4 99.5 FeCl 3 98.1 FeSO 4 96.6 HgCl 2 5.9 MgCl 2 101.5 MnCl 2 100.9 NiCl 2 93.4 Pb(OAc) 2 99.8 ZnSO 4 102.1 Chemical Concn.(mM) Residual activity(%) KF 2.0 98.7 NaF 10.0 100.6 NaN 3 20.0 101.6 NEM 2.0 97.6 MIA 2.0 0.4 IAA 2.0 92.2 EDTA 5.0 107.2 (NH 4 ) 2 SO 4 20.0 96.0 Borate 20.0 101.4 o-Phenanthroline 2.0 97.7 α,α′-Dipyridyl 1.0 100.3 Urea 2.0 122.5 Guanidine 2.0 99.2 Hydroxylamine 2.0 107.2 Ac, CH 3 CO; NEM, N-Ethylmaleimide; MIA, Monoiodoacetate; IAA, lodoacetamide; EDTA, Ethylenediaminetetraacetate. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. Stability (Powder form) (kept under dry conditions) Fig.3. Stability (Liquid form) 25 ℃,in 83 mM Tris-HCI buffer solution pH 8.0(contg.3.7 mM β-NAD,40 U/mL mutarotase) enzyme concn.:300 U/mL Fig.4. pH-Activity 37 ℃,5 min-reaction in 80 mM buffer solution ●:pH 6.0-8.0 K-phosphate ○:pH 8.0-9.0,Tris-HCI ■:pH 8.5-10.5 Carbonate Fig.5. Temperature activity (in 80 mM Tris-HCI buffer, pH 8.0) Fig.6. pH-Stability 20 ℃,16 hr with 0.1 M buffer solution ●:pH 4.0-6.0 acetate ○:pH 6.0-8.0 K-phosphate ■:pH 7.5-9.0 Tris-HCI □:pH 8.5-10.5 carbonate enzyme concn.:10 U/mL Fig.7. Thermal stability 15 min-treatment with 50 mM K-phosphate buffer pH 7.0 enzyme concn.: 12 U/mL