GLUCOSE OXIDASE from Aspergillus sp.

PREPARATION and SPECIFICATION Appearance Yellowish amorphous powder, lyophilized Activity GradeⅠ 180 U/mg-solid or more GradeⅡ 100 U/mg-solid or more (containing approx. 50 % of stabilizers) Contaminant Catalase GradeⅠ ≤ 5.0×10 -3  % GradeⅡ ≤ 3.0 % Stabilizers Potassium gluconate, sodium glutamate PROPERTIES Stability Stable at −20 ℃ for at least one year (Fig.1) Molecular weight approx. 153,000 Michaelis constants 3.3×10 -2  M (β-D-Glucose), 3) 6.1×10 -2  M (2-Deoxyglucose) Structure Glycoprotein with 2 moles of FAD Inhibitors p-Chloromercuribenzoate, heavy metal ions (Cu 2＋ , Hg 2＋ , Ag ＋ ) Optimum pH 4.5 (Fig.3) Optimum temperature 40 − 50 ℃ (Fig.4) pH Stability pH 4.5−6.0 (30 ℃, 20 hr) (Fig.5) Thermal stability below 50 ℃ (pH 5.7, 1 hr) (Fig.6) Substrate specificty (Table 1) Effect of various chemicals (Table 2) APPLICATIONS This enzyme is useful for enzymatic determination of glucose, and for an amylase-activity assay in combination with α-glucosidase ( AGH-211 , if maltooligosaccharide or modified starch is used as a substrate), in clinical analysis. ASSAY Principle 4-AA : 4-Aminoantipyrine EHSPT : N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine The formation of quinoneimine dye is measured at 555 nm by spectrophotometry. Unit definition One unit catalyzes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions detailed below. Method Reagents A. MES-Na buffer pH 5.7 0.1 M: Dissolve 2.13 g of 2-(N-morpholino) ethansulfonic acid (MW ＝ 213.25) in approx. 60 mL of H 2 O and, after adjusting the pH to 5.7 with 1 N NaOH at 25℃, make up to 100 mL with H2O (stable at 5℃ for 1 month). B. Glucose solution 15 %: Dissolve 1.5 g of β-D-glucose in H 2 O, and make up to 10 mL, at least 2 hours before, but on the same day as, the assay. C. 4-AA solution 0.5 %: 50 mg of 4-aminoantipyrine (MW ＝ 203.25) / 10 mL of H 2 O (stable at 5℃ in a brownish bottle for at least 1 week). D. EHSPT solution 40 mM: 118 mg of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (MW ＝ 295.3) / 10 mL of H 2 O (stable at 5℃ in a brownish bottle for at least 1 week). E. Peroxidase solution 500 purpurogalin units (U) /mL H 2 O F. Enzyme diluent 10 mM MES-Na buffer, pH 5.7, containing 0.1 % Triton X-100 Procedure 1.Prepare the following working solution in a brownish bottle and store on ice. (Should be prepared fresh) 30 mL Buffer solution (A) 6.0 mL Substrate solution (B) 0.3 mL 4-AA solution (C) 0.3 mL EHSPT solution (D) 0.3 mL POD solution (E) Concentration in assay mixture MES buffer 79 mM D-glucose 131 mM 4-AA 0.2 mM EHSPT 0.3 mM POD ca.4 U/ml 2.Pipette 3.0 mL of working solution into a cuvette (d ＝ 1.0cm) and equilibrate at 37 ℃ for approximately 5 minutes. 3.Add 0.1 mL of the enzyme solution *  and mix by gentle inversion. 4.Record the increase in optical density at 555 nm against water for 2 to 3 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 enzyme diluent (F) is added instead of the enzyme solution. * Dissolve the enzyme preparation in ice cold enzyme diluent (F) and dilute to 0.05−0.2 U/mL with the same buffer, immediately before the assay. Calculation Activity can be calculated by using the following formula : Volume activity (U/mL) = ΔOD/min (ΔOD test−ΔOD blank)×Vt×df 32.8×1/2×1.0×Vs ＝ ΔOD/min×1.89×df Weight activity (U/mg) ＝ (U/mL)×1/C Vt : Total volume (3.1 mL) Vs : Sample volume (0.1 mL) 32.8 : Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm 2 /micromole) 1/2 : Factor based on the fact that one mole of H 2 O 2  produces a half of quinoneimine dye. 1.0 : Light path length (cm) df : Dilution factor C : Enzyme concentration in dissolution (c mg/mL) REFERENCES 1)The Enzymes, Vol.ⅫB, P.421 (P.D.Boyer, ed.), Academic Press (1975). 2)Method in Enzymology, Vol.Ⅸ, p.82 (S.P.Colowick and N.O.Kaplan, ed.),Academic Press (1966). 3)B.E.P.Swoboda and V.Massay; J.Biol.Chem., 240, 2209 (1965). 4)P.J.Auses, S.L.Cook and J.T.Maloy; Anal.Chem., 47, 244 (1975). 5)D.C.Williams, G.F.Huff and W.R.Gaitz; Clin.Chem., 22, 372 (1976). Table 1. Substrate Specificity of Glucose oxidase [0.1M of Substrate, 79mM MES buffer, pH 5.7, at 30 ℃ ] Substrate (0.1M) Relative activity(%) D-Glucose 100 2-Dexy-D-glucose 16.2 Glucono-1,5-lactone 0.06 L-Glucose 0.00 Galactose 3.10 Mannose 2.10 Substrate (0.1M) Relative activity(%) Fructose 0.24 Xylose 0.93 Ribose 0.00 Maltose 0.69 Lactose 0.00 Table 2. Effect of Various Chemicals on Glucose oxidase ［The enzyme dissolved in 0.1M MES buffer, pH 5.7 (10 U/mL) was incubated with each chemical for 2 hr at 25 ℃.］ Chemical Concn.(mM) Residual activity(%) None - 100 Metal salt 2.0 MgCl 2 92.6 CaCl 2 93.6 BaCl 2 94.4 CoCl 2 98.1 MnCl 2 95.1 ZnSO 2 94.3 FeCl 2 96.8 NiCl 2 91.7 CuSO 4 71.6 AgNO 3 58.6 HgCl 2 0.7 PCMB 2.0 31.6 MIA 2.0 96.8 NaF 2.0 97.1 Chemical Concn.(mM) Residual activity(%) NaN 3 20 96.3 EDTA 5.0 97.3 o-Phenanthroline 2.0 95.3 α,α′-Dipyridyl 2.0 99.5 Borate 50.0 96.1 IAA 2.0 96.1 MIA 2.0 101.1 Hydroxylamine 10.0 98.3 Sodium bisulfite 10.0 100.0 hydrazine 10.0 103.1 Triton X-100 0.1 % 111.2 Brij 35 0.1 % 108.0 Tween 20 0.1 % 110.7 Span 20 0.1 % 106.7 Na-Cholate 0.1 % 106.1 SDS 0.1 % 113.1 PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediamimetetraacetate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate. Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. Stability (Powder form) (kept under dry conditions) Fig.3. pH-Activity 37 ℃, 5min-reaction in 79 mM buffer solution : ○̶̶○ . acetate;●̶̶● MES;△̶̶△, BES;×̶̶×, BICINE Fig.4. Temperature activity (5 min-reaction in 79 mM MES buffer, pH5.7) Fig.5. pH-Stability 30 ℃, 20hr-treatment with 0.1 M buffer solution : ○̶̶○ . acetate : ●̶̶● MES : △̶̶△, BES : ×̶̶×, BICINE Fig.6. Thermal stability (1hr-treatment in 79 mM MES buffer , pH5.7)