GLUCOSE DEHYDROGENASE (PQQ-dependent) from Microorganism

PREPARATION and SPECIFICATION Appearance Purple amorphous powder, lyophilized Activity Grade Ⅲ　500 U/mg-solid or more Contaminants Glucose dehydrogenase (NAD-dependent) ≤ 1.0×10 -3  % Hexokinase ≤ 1.0×10 -3  % Stabilizers Ca 2＋ , BSA PROPERTIES Stability Stable at − 20℃ for at least one year （Fig.1） Molecular weight approx. 100,000 (by gel filtration) Michaelis constant 4.8 mM (D-Glucose) Inhibitors Cu 2＋ , Pb 2＋ , Ag ＋ Optimum pH 7.0 （Fig.2） Optimum temperature 37 ℃ （Fig.3） pH Stability pH 3.5−8.5 (25 ℃, 16 hr) （Fig.4） Thermal stability below 50 ℃ (pH 7.5, 30 min) （Fig.5） Substrate specificity (Table 1) Effect of various chemicals (Table 2) APPLICATIONS This enzyme is useful for enzymatic determination of D-glucose. ASSAY Principle The formation of diformazan formed by the reduction of nitrotetrazorium blue (NTB) with phenazine methosulfate (PMS), which is red, is measured at 570 nm by spectrophotometry. Unit definition One unit causes the formation of one half micromole of diformazan per minute under the conditions detailed below. Method Reagents A. D-Glucose solution 1 M: 1.8 g of D-glucose (MW ＝ 180.16) / 10 mL of H 2 O (keep this solution at room temperature for at least 3 hours before use). B. PIPES-NaOH buffer, pH 6.5 50 mM: Weigh out 1.51 g of PIPES (MW ＝ 302.36), suspend in 60 mL of H 2 O, dissolve with 5 N NaOH, and add 2.2 mL of 10 % Triton X-100. After adjusting pH to 6.5 ± 0.05 at 25℃ with 5 N NaOH, make up to 100mL with H 2 O. C. PMS solution 3.0 mM: 9.19 mg of phenazine methosulfate (MW ＝ 306.34) / 10 mL of H 2 O D. NTB solution 6.6 mM: 53.96 mg of NTB (MW ＝ 817.65) / 10 mL of H 2 O E. Enzyme diluent 50 mM PIPES-NaOH buffer, pH 6.5, containing 1 mM CaCl 2 , 0.1 % Triton X-100, 0.1 % BSA Procedure 1.Prepare the following reaction mixture in a brownish bottle shortly before use, and store on ice. 0.9 mL D-glucose solution (A) 25.5 mL PIPES-NaOH buffer, pH 6.5 (B) 2.0 mL PMS solution (C) 1.0 mL NTB solution (D) Concentration in assay mixture PIPES-buffer 42 mM D-glucose 30 mM PMS 0.20 mM NTB 0.22 mM 2.Pipette 3.0 mL of working solution into a plastic test tube and equilibrate at 37 ℃ for approximately 5 minutes. 3.Add 0.1 mL of enzyme solution* and mix by gentle inversion. 4.Record the increase of optical density at 570 nm against water for 4 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) by the same method as in the test except that the enzyme diluent (E) is added instead of the enzyme solution. * Dissolve the enzyme preparation on ice cold enzyme diluent (E) and dilute to 0.1−0.8 U/mL with the same buffer, immediately before the assay. (A plastic tube is recommended because of viscous properties of the liquid.) Calculation Activity can be calculated by using the following formula : Volume activity (U/mL) = ΔOD/min (ΔOD test−ΔOD blank)×Vt×df 20.1×1.0×Vs ＝ ΔOD×1.54×df Weight activity (U/mg) ＝ (U/mL)×1/C Vt : Total volume (3.1 mL) Vs : Sample volume (0.1 mL) 20.1 : Half a millimolar extinction coefficient of diformazan (cm 2 /0.5 micromole) 1.0 : Light path length (cm) df : Dilution factor C : Enzyme concentration in dissolution (c mg/mL) REFERENCES 1)K.Matsushita et al.; FEMS Microbiology Letters, 55, 53 (1988). Table 1. Substrate Specificity of PQQ-Glucose dehydrogenase Substrate (50mM) Relative activity(%) D-Glucose 100.0 L-Glucose 0.3 D-Xylose 15.0 2-Deoxy-glucose 4.9 L-Sorbose 0.5 D-Mannose 10.8 D-Fructose 0.3 Substrate (50mM) Relative activity(%) Galactose 16.0 D-Lactose 68.9 D-Sorbitole 0.2 D-Mannitol 0.0 Sucrose 0.2 Inositol 0.0 Maltose 107.0 Table 2. Effect of Various Chemicals on PQQ-Glucose dehydrogenase ［The enzyme dissolved in 50mM PIPES-NaOH buffer, pH 6.5 contg. 1mM CaCl 2 , 0.1% Triton X-100 (5U/mL) was incubated with each chemical at 25℃ for 1hr.］ Chemical Concn.(mM) Residual activity(%) None - 100 Metal salt 2.0 MgSO 4 108 CaCl 2 108 Ba(OAc) 2 105 FeCl 3 79 CoCl 2 42 MnCl 2 105 ZnCl 2 45 Cd(OAc) 2 107 NiCl 2 101 CuSO 4 0 Pb(OAc) 2 0 AgNO 3 0 HgCl 2 77 2-Mercaptoethanol 2.0 99 PCMB 1.0 97 Chemical Concn.(mM) Residual activity(%) MIA 2.0 87 NEM 2.0 100 IAA 2.0 98 Hydroxylamine 2.0 19 EDTA 5.0 79 O-Phenanthroline 2.0 7 α,α′-Dipyridyl 1.0 103 Borate 5.0 110 NAF 2.0 111 NaN 3 2.0 115 Triton X-100 0.10 % 101 Brij 35 0.10 % 22 Tween 20 0.10 % 104 Span 20 0.10 % 60 Na-Cholate 0.10 % 67 SDS 0.05 % 33 DAC 0.05 % 113 Ac, CH 3 CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. pH-Activity 25℃, in 50mM buffer solution; ●̶●,acetate; ▼̶▼, phosphate; ○̶○, PIPES; ▽̶▽, Tris-HCl. Fig.3. Temperature Activity (in 42mM PIPES-NaOH buffer, pH 6.5) Fig.4. pH-Stability 25℃, 16 hr-treatment with 50mM buffer solution contg. 1mM CaCl2; ●̶●,acetate; ▼̶▼, phosphate; ○̶○, PIPES; ▽̶▽, Tris-HCl. Fig.5. Thermal stability 30min.-treatment with 50mM PIPES-NaOH buffer, pH 6.5 contg. 1mM CaCl 2  enzyme concentration: 5.0 U/mL