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alpha-Glucosidase

(a-D-Glucoside glucohydrolase; EC 3.2.1.20)

Alpha-glucosidase is also known as maltase. It catalyzes the hydrolysis of maltose to glucose units. Mammalian intestinal mucosa secretes disaccharidases such as maltose, lactose and sucrose. Alpha-glucosidase (maltase) is used for assaying the activity of alpha amylase and for the determination of maltose in brewing.
Alpha-glucosidase catalyzes the following reaction:

a-GLUCOSIDASE

a-D-Glucoside + H2O D-Glucose + Alcohol






The amount of enzyme which catalyzes the release of one micromole of glucose per minute at 25° C, pH 6.0.



The increase in absorbance at 340 nm, caused by the reduction of NADP is a measure of the catalytic activity of a-Glucosidase.



1. 0.1 M Acetate buffer, pH 6.0; 1.35 mM EDTA. Dissolve 50 mg EDTA-Na2• H2O in 0.57 ml acetic acid (96%)/80 ml redistilled water. Adjust pH to 6.0 with 1 M NaOH. Adjust volume to 100 ml with redistilled water.
2. 0.56 M Maltose• 1H2O in 100 ml redistilled water.
3. 0.3 M Triethanolamine buffer, pH 7.6. Dissolve 5.58 g TEA/HCl in 80 ml redistilled water. Adjust to pH 7.6 with 1 M NaOH. Adjust volume to 100 ml with redistilled water.
4. 0.1 M Magnesium Chloride. Dissolve 2.03 g MgCl2• 6 H2O in 100 ml redistilled water.
5. 17 mM ATP. Dissolve 10 mg ATP-Na in 1 ml redistilled water.
6. 13 mM NADP. Dissolve 10 mg NADP-Na2 in 1 ml redistilled water.
7. Glucose-6-phosphate dehydrogenase from yeast: 1 mg protein/ml.
8. Hexokinase from yeast: 2 mg protein/ml.
9. a-Glucosidase (enzyme) solution: For freeze-dried enzyme, dissolve 10 mg in 12 ml cold redistilled water. For enzyme suspension, use a ration of 1:50 with cold redistilled water. Prepare the enzyme sample just prior to measurement. Volume activity should be 5 U/ml.



1. Set up boiling water bath and spectrophotometer at 340 nm.
2. For incubation, prepare a blank and a sample in two centrifuge tubes. Pipette the following into each tube:
Acetate buffer 1.5 ml
Maltose 0.5 ml
Sample 0.05 ml
3. Mix and incubate at 25° C for exactly 5 min., then add sample to each and place both tubes into boiling waterbath for 3 min.
4. Remove from waterbath and centrifuge for 5 min. at 3000 rpm.
5. Use 0.1 ml of these pretreated blank and sample solutions for the assay mixture.
6. Pipette the following into each cuvette, blank and sample:
TEA buffer 2.5 ml
MgCl2 0.1 ml
ATP 0.1 ml
NADP 0.1 ml
G-6-PDH 0.01 ml
7. Add 0.1 ml of redistilled water to the blank and 0.1 ml of the enzyme solution to the sample cuvette; mix and read absorbance (E340nm/min).
8. Start reaction by addition of 0.03 ml Hexokinase to each cuvette. Mix and read absorbance (ÆE340nm/min - BLANK).
9. Calculate E340nm/min.



Activity (U/mg) = (ΔE340nm/min2 - ΔE340nm/min1)(Total Vol.)(Enz. Diln.)
(6.22)(Enz. Vol.)(mg Enz./ml)
 

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