GLUCOAMYLASE from Rhizopus sp.


Appearance White amorphous powder (salt-free), lyophilized
Activity GradeⅠ 30U/mg-solid or more
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx. 70,000 ¹⁾
Michaelis constants¹ ⁾ 11±1.1×10⁻⁴M (Maltose), 3.6±0.51×10⁻⁴M (Maltotriose),
2.5±0.33×10⁻⁴M (Maltotetraose), 1.6±0.02×10⁻⁴M (Maltopentaose)
Optimum pH 4.5-5.0(Fig.3)
Optimum temperature 60℃(Fig.4)
pH Stability pH 4.0-8.5 (25℃, 20hr)(Fig.5)
Thermal stability  below 45℃ (pH 5.5, 10min)(Fig.6)
Substrate specificty¹· ² ⁾ This enzyme completely hydrolyzes soluble starch, amylopectin,
glycogen,α-orß-limit dextrin, amylose, maltooligosaccharides and panose.


This enzyme is useful for structural investigation of carbohydrates and for enzymatic determination of α-amylase when coupled with the related enzymes in clinical analysis.




Starch+n H₂O                                  ►n Glucose+Dextrin

The formation of glucose is measured as reducing sugar 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 described below.


A. Starch solution 1.0%[Suspend 1.0g of soluble starch (Merck) in 90ml of HO, dissolve by boiling for 3min and cool down to room temperature. Add 5.0ml of 1.0M acetate buffer, pH 4.5 and fill up to 100ml with HO.] (Should be prepared fresh)
B. Alkaline solution 100g NaOH, 365g Rochelle salt・4HO/1,000ml of HO
C. CuSO₄ Solution 7.0% (70g CuSO₄・5HO/1,000ml of HO)
D. KI solution 30% (300g KI/1,000ml of HO)(Store in a brownish bottle)
E. HSO₄ Solution 25%
F. NaSO₃ Solution 50mM (49.638g NaSO₃ ・5HO, 4.0g NaCO₃ (stabilizer)/4,000ml of HO)
(Store in a brownish bottle and keep for 3~4 days before use)
G. Enzyme diluent 10mM acetate buffer, pH 4.5


Concentration in assay mixture
Acetate buffer 42 mM
Starch 0.8%

1. Pipette 4.0ml of substrate solution (A) into a test tube (32ø× 200mm) and equilibrate 40℃ for about 5minutes.

2. Add 1.0ml of the enzyme solution* and mix

3. After exactly 15 minutes at 40℃, add 2.0ml of alkaline solution (B) stop the reaction.
At the same time, prepare the blank by first mixing the substrate solution with 2.0ml of alkaline solution after 15min-incubation at 40℃, followed by addition of the enzyme solution.

4. Add 2.0ml of CuSO₄ solution (C) and, after covering the test tube with a marble (40mmø) to prevent evaporation, place the test tube in a boiling water bath.

5. After 20 minutes, remove the test tube from a boiling water bath and cool down to room temperature under running water.

6. Add 2.0ml each of KI solution (D) and HSO₄ solution (E) in this order.

7. Shake the test tube and determine the amount of residual Cu⁺⁺ by titration with NaSO₃ solution (F).

8. Record the titers (ml) of the test (Δt) and the blank (Δb), and calculate the titration difference in ml (Δsample:Δb-Δt).

* Dissolve the enzyme preparation in ice-cold distilled water and dilute to 0.4-1.5U/ml with enzyme diluent
(G), immediately before assay.


Activity can be calculated by using the following formula :

Weight activity (U/mg)=(U/ml)×1/C

: 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.)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  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).


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