GLUCOAMYLASE from Rhizopus sp.

PREPARATION and SPECIFICATION Appearance White amorphous powder (salt-free), lyophilized Activity GradeⅠ　30 U/mg-solid or more PROPERTIES Stability Stable at −20 ℃ for at least one year （Fig.1） Molecular weight approx. 70,000 1) Michaelis constants 1) 11 ± 1.1×10 -4  M (Maltose), 3.6 ± 0.51×10 -4  M (Maltotriose), 2.5 ± 0.33×10 -4  M (Maltotetraose), 1.6 ± 0.02×10 -4  M (Maltopentaose) Structure Glycoprotein［E  280 nm 1 cm  (1 %)＝14.5］ 1 cm Optimum pH 4.5−5.0 （Fig.3） Optimum temperature 60 ℃ （Fig.4） pH Stability pH 4.0−8.5 (25 ℃, 20 hr) （Fig.5） Thermal stability below 45 ℃ (pH 5.5, 10 min) （Fig.6） Substrate specificity 1,2) This enzyme completely hydrolyzes soluble starch, amylopectin, glycogen,α-orβ-limit dextrin, amylose, maltooligosaccharides and panose. APPLICATIONS This enzyme is useful for structural analysis of carbohydrates and for enzymatic determination of α-amylase in combination with the related enzymes in clinical analysis. ASSAY Principle The formation of glucose is measured with reducing sugar as the index, by the modified Fehling-Lehmann-Schoorl method. Unit definition One unit causes the formation of ten milligrams of glucose in 30 minutes under the conditions detailed below. Method Reagents A. Starch solution 1.0 %: Suspend 1.0 g of soluble starch (Merck) in 90 mL of H 2 O, dissolve by boiling for 3 min, and cool to room temperature. Add 5.0 mL of 1.0 M acetate buffer, pH 4.5, and make up to 100 mL with H 2 O (should be freshly prepared). B. Alkaline solution 100 g of NaOH, 365 g of potassium sodium tartrate tetrahydrate, per 1,000 mL of H 2 O C. CuSO 4  Solution 7.0 % :70 g CuSO 4 ・5H 2 O/1,000 mL of H 2 O D. KI solution 30 %: 300 g of KI per 1,000 mL of H 2 O (store in a brownish bottle) E. H 2 SO 4  Solution 25 % F. Na 2 S 2 O 3  Solution 50 mM: 49.638 g pf Na 2 S 2 O3・5H 2 O, 4.0 g of Na 2 CO 3  (stabilizer) in 4,000 mL of H 2 O (store in a brownish bottle and keep for 3～4 days before use). G. Enzyme diluent 10 mM acetate buffer, pH 4.5 Procedure 1.Pipette 4.0 mL of substrate solution (A) into a test tube (32 φ× 200 mm) and equilibrate at 40 ℃ for approximately 5minutes. Concentration in assay mixture Acetate buffer 42 mM Starch 0.8 % 2.Add 1.0 mL of the enzyme solution* and mix. 3.After exactly 15 minutes at 40 ℃, add 2.0 mL of alkaline solution (B) to stop the reaction. At the same time, prepare the blank by first mixing the substrate solution with 2.0 mL of alkaline solution after incubation for 15 min at 40 ℃, followed by addition of the enzyme solution. 4.Add 2.0 mL of CuSO 4  solution (C) and, after covering the test tube with a marble (40 mmφ) to prevent evaporation, place the test tube in a boiling water bath. 5.After 20 minutes, remove the test tube from the boiling water bath and cool to room temperature under running water. 6.Add 2.0 mL each of KI solution (D) and H 2 SO 4  solution (E) in this order. 7.Shake the test tube and determine the amount of residual Cu 2＋  by titration with Na 2 S 2 O 3  solution (F). 8.Record the titers (mL) of the test (Δt) and the blank (Δb), and calculate the titration difference in mL (Δ sample: Δb−Δt). * Dissolve the enzyme preparation in ice-cold distilled water and dilute to 0.4−1.5 U/mL with enzyme diluent (G), immediately before assay. Calculation Activity can be calculated by using the following formula : Volume activity (U/mL) = Δsample×30 min×df Δglucose×15 min ＝ Δsample Δglucose ×2.0×df Weight activity (U/mg) ＝ (U/mL)×1/C Δglucose : Titration difference (mL) for ten miligrams of glucose (Determine the titration difference by using glucose standard solution (5.0mg/mL) instead of the enzyme solution under the above assay conditions.) df : Dilution factor C : Enzyme concentration in dissolution (c mg/mL) REFERENCES 1)K.Hiromi, Y. Nitta, C.Numata and S.Ono; Biochim.Biophys.Acta, 302, 362 (1973). 2)J.Fukumoto; Protein, Nucleic Acid and Enzyme, 4, 3 (1959). Fig.1. Stability (Powder form) (kept under dry conditions) Fig.2. Stability (Powder form) (kept under dry conditions) Fig.3. pH-Activity 40℃,15min-reaction in 50mM buffer solution: pH2.0,sodium acetate-HCI; pH3.0-6.0,acetate;pH6.0-7.0, phosphate Fig.4.Temperature activity 15min-reaction in 50mM acetate buffer,pH4.5 Fig.5.pH-Stability 25℃,20hr-treatment with 50mM buffer solution: pH3.0-6.0 acetate; pH6.0-9.0,phosphate;pH9.0-10.0, borate