Description
FAD-dependent glucose dehydrogenase is an enzyme used as a regeneration cofactor to convert glucose and NAD(P) into NAD(P)H and gluconic acid.ORIGIN: Aspergillus oryzae CATALYSIS: SPECIFICATIONSActivity≥100 U/mg powderAppearanceYellow powder, lyophilizedStabilityStable for 36 months at -20 ℃AdditivesNot AddedCHARACTERISTICSMolecular weight180,000 (Gel filtration)Isoelectric point6.5 Km (Glucose)15.0 × 10⁻³ M InhibitorsAg⁺, Hg²⁺ ActivatorTriton X-100 Opt. pH7.0Fig.1Opt. temperature55 ℃Fig.2Stable pH range4.0-7.0Fig.3Stable temp. rangebelow 40 ℃Fig.4Substrate specificity Table 1Mediator Preference Fig.5APPLICATIONS This enzyme is used for enzymatic determination of glucose in blood or urine by glucose sensor etc.Assay Method of Glucose Dehydrogenase (GDH-FAD)Principle1* phenadine methosulfate, 2* Nitorotetrazorium blueThe appearance of diformazan is measured at 570nm by spectrophotometry.Unit Definition One unit is defined as the enzyme quantity which produces 0.5 µmol of diformazan per minute under the conditions described below.ReagentsA. Triton X-100 Solution (5% Triton X-100 solution)Weigh 5.0 g of Triton X-100 and dissolve in 30 mL of deionized water with heating. After cooling, fill up to 100 mL with deionized water. (Expires after 1 month at room temperature)B. Working Solution (50 mmol/L PIPES-NaOH (pH 6.5) containing 0.5 % Triton X-100)Weigh 1.51 g of PIPES and dissolve in approx. 70mL of deionized water. Add 10 mL of Triton X-100 solution (A), then adjust the pH to 6.5 with 4N NaOH. Fill up to 100 mL with deionized water. (Expires after 1 month at 2~8℃)C. NTB Solution (6.6 mmol/L)Weigh 54 mg of Nitorotetrazorium blue and dissolve in 10 mL of deionized water. This solution should be kept in a light-proof tube to avoid exposure to light. (Expires after 14 days at 2~8℃)D. PMS Solution (3.0 mmol/L)Weigh 9 mg of phenazine methosulfate and dissolve in 10 mL of deionized water. This solution should be kept in a light-proof tube to avoid exposure to light. (Expires after 14 days at 2~8℃)E. Substrate Solution (1.0 mol/L Glucose)Weigh 3.6 g of D-glucose and dissolve in deionized water. Fill up to 20 mL with deionized water. (Expires after 14 days at room temperature)F. Enzyme DiluentWeigh 3.02 g of PIPES and 0.2 g of bovine serum albumin and 29.4 mg of CaCl₂·2H₂O in approx. 160 mL of deionized water. Add 4 mL of Triton X-100 solution (A), then adjust the pH to 6.5 with 4N NaOH. Fill up to 200 mL with deionized water. (Expires after 1 month at 2~8℃)G. Enzyme SolutionWeigh 25 mg of Glucose Dehydrogenase (GDH-FAD) and dissolve in chilled Enzyme Diluent (F). Enzyme Solution should be prepared so that the value of ΔOD/2minutes becomes in the range of 0.058±0.026.H. Mix SolutionMix 26 mL of Working Solution (B), 1 mL of NTB Solution (C), 2 mL of PMS Solution (D) and 1 mL of Substrate Solution (E). This solution should be kept in a light-proof tube to avoid exposure to light. (Expires 6 hours at 2~8℃)ProcedurePipette 3 mL of Mix Solution (H) into a disposable plastics cuvette (d=10 ~mm) and keep at 37±0.5 ℃ for 10 minutes. Then, pipette 0.1 mL of Enzyme Solution (E) into the cuvette and mix well immediately. Keep the reaction mixture at 37±0.5 ℃. Exactly at 3 minutes and 5 minutes after the addition of Enzyme Solution (E), measure the absorbances of the reaction mixture at 570 nm. (A3 and A5) As a blank, pipette Enzyme Diluent (F) into another disposable plastics cuvette (d=10 ~mm) instead of Enzyme Solution (E), and take the same procedure described above. (Ab3 and Ab5)Calculation2: Reaction time20.1: Half of millimolar extinction coefficient of diformazan at 570 nm3.1: Final volume of the reaction mixture0.1: Volume of Enzyme SolutionDm: Dilution multiple of Enzyme Solution