GLS-A (CD: 60126)

-Glucosidase ( GLS-A)
from sweet almond
-D-Glucoside glucohydrolase, EC 3.2.1.21
-D-Glucoside  H2O -D-Glucose  ROH

SPECIFICATION
Appearance
Activity
Stabilizer
Storage
PROPERTIES
Molecular weight
Structure
Michaelis constants
pH Optimum
pH Stability
Optimum temperature
Thermal stability
Stability (liquid form)
Stability (powder form)

white lyophilizate
10 U/mg lyophilizate
bovine serum albumin
at 20C

ca. 98 kDa (gel filtration)

2 subunits of 49 kDa (SDS-PAGE)
2.6103 M ( p-nitrophenyl--D-glucoside)
2.4103 M (2-chloro-4-nitrophenyl--D-glucoside)
5.05.5
(Fig. 1)
4.08.0
(Fig. 2)
5055C
(Fig. 3)
below 50C
(Fig. 4)
stable at 37C for at least two weeks
(Fig. 5)
stable at 30C for at least one month
(Fig. 6)

81

 GLS-A (CD: 60126)

ASSAY PROCEDURE
Principle

-glucosidase
p-Nitrophenyl--D-glucoside (PNPG) p-Nitrophenol (PNP)  D-Glucose
The appearance of p-nitrophenol is measured spectrophotometrically at 400 nm.
Definition of unit
One unit (U) is defined as the amount of enzyme which produces 1 mol of PNP per min at 37C and pH 5.0 under
the conditions described below.
Reagents
A. TrisHCl buffer, 50 mM; pH 7.8: dissolve 0.61 g Tris(hydroxymethyl)aminomethane in 80 ml of distilled water,
adjust to pH 7.8 with 1 N HCl and dilute with distilled water to 100 ml.
B. Potassium phosphate buffer, 10 mM; pH 7.0, containing bovine serum albumin (BSA): mix 10 mM KH2PO4
solution and 10 mM K2HPO4 solution to make a pH 7.0 solution and add BSA (0.2 g/100 ml of the buffer).
C. p-Nitrophenyl--D-glucoside (PNPG) solution, 20 mM: 60.3 mg of PNPG/10 ml of distilled water.
D. Acetate buffer, 0.1 M; pH 5.0: mix 0.1 M sodium acetate solution and 0.1 M acetic acid solution to make a pH 5.0
solution.
E. Sodium carbonate solution, 0.2 M: 2.12 g of Na2CO3 (anhydrous)/100 ml of distilled water.
Sample: dissolve the lyophilized enzyme in ice-cold TrisHCl buffer (Reagent A) and dilute to a volume activity of
0.0060.018 U/ml with ice-cold potassium phosphate buffer (Reagent B) immediately before measurement.
Procedure
1. Pipette the following reagents into a test tube.
0.5 ml PNPG solution (Reagent C)
1.0 ml Acetate buffer
(Reagent D)
2. Equilibrate at 37C for about 5 min.
3. Add 0.5 ml of sample and incubate for 15 min at 37C.
4. Add 2.0 ml of sodium carbonate solution (Reagent E) to stop the reaction.
5. Read the absorbance at 400 nm in a cuvette (light path: 1 cm) (AS).
The blank solution is prepared by reversing the sequence of addition of sample and sodium carbonate solution
(Reagent E) (A0).

82

 GLS-A (CD: 60126)

Calculation
Activity can be calculated by using the following formula:
Volume activity (U/ml) 

( ASA0 )  4.0 ( ml)  df

 A  0.0295  df
18.1 0.5 (ml) 15 (min)

Weight activity (U/mg)  (U/ml)  1/C

18.1 : Millimolar extinction coefficient of PNP under the assay conditions (cm2/mol)
df : Dilution factor
C : Content of -glucosidase preparation in sample (mg/ml)

APPLICATIONS
The enzyme is useful for the determination of -amylase in clinical analysis.

-amylase
N3G5-CNP N3G3  G2-CNP
glucoamylase
G2-CNP G  G1-CNP

-glucosidase
G1-CNP G  CNP
(N3G5-CNP: 2-chloro-4-nitrophenyl 65-azido-65-deoxy--maltopentaoside)

REFERENCE
Grover, A. K. et al., Biochim. Biophy. Acta, 482, 98108 (1977).

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 GLS-A (CD: 60126)

EXPERIMENTAL DATA
Fig. 2 pH Stability

Fig. 1 pH Optimum
100

Residual activity (%)

Relative activity (%)

100
80
60
40
20
0

80
60
40
20
0

pH

pH
Treatment: 25C, 20 h

: 50 mM acetate buffer
: 50 mM -glycerophosphate buffer

: 50 mM acetate buffer
: 50 mM TrisHCl buffer
: 50 mM -glycerophosphate buffer

Fig. 4 Thermal stability

Fig. 3 Optimum temperature

100

Residual activity (%)

Relative activity (%)

100
80
60
40
20
0

80
60
40
20
0

20

30

40

50

60

70

80

20

30

40

50

60

70

C
Treatment: 1 h

: 50 mM acetate buffer, pH 5.0

: 50 mM -glycerophosphate buffer, pH 6.8

: 50 mM acetate buffer, pH 5.0

: 50 mM TrisHCl buffer, pH 7.3

Fig. 5 Stability (liquid form) at 37C

Fig. 6 Stability (powder form) at 30C

100

Residual activity (%)

Residual activity (%)

100
80
60
40
20
0

60
40
20
0

84

80

12

15

Day

Week

(Kept in 50 mM Tris-HCl buffer, pH 7.5)

(Kept under dry conditions)