Glucoamylase activity assay

I. Introduction
Glucoamylase is one of the most widely used and oldest enzymes in the food industry.
The major application of this enzyme is in the saccharification of starch/dextrin to
glucose, which is an essential substrate for numerous fermentation processes.
Glucoamylase could hydrolyze both α-1,4- and α-1,6 glycosidic linkages in starch but
the hydrolysis occurs at a comparatively slower rate for the α-1,6 glycosidic bond. In
this procedure, glucoamylase activity is determined based on the amount of free glucose
released after hydrolysis of soluble starch, which could be quantified by a reducing
sugar method, in this Lab 6, DNS method will be used.
II. Materials
- 1% starch solution
- Enzyme solution (at appropriate dilution)
- DNS reagent
- Distilled water
- Micropipettes, water bath.
III. Assay Procedure
- Pre-equilibrate enzyme and starch solution at 30°C for at least 5 min.
- Pipette the following reagent into glass tubes
Test sample Blank sample
Enzyme - 0.5 mL of starch solution. - Add 3 mL of DNS to the tube.
reaction - 0.5 mL of enzyme solution. - Add 0.5 mL of enzyme solution.
- Mix well (Votex). - Mix well by vortex.
- Incubate exactly 10 minutes. - Incubate for 10 minutes.
Enzyme - Add immediately 3 mL of DNS. - Add 0.5 mL of starch solution.
inactivation - Mix well by vortex. - Mix well by vortex
by DNS
Glucose - Incubate the tubes in a boiling water bath for 5 minutes.
quantification - Cool the tubes by tape water.
by DNS
Measuring - Measure the absorbance at 540 nm (use the blank sample to set the
absorbance spectrophotometer to zero).
value
 IV. Calculation
Calculate the glucoamylase activity (U/mL) using the DNS-glucose standard curve
previously established.
One unit of glucoamylase activity is defined as the amount of enzyme which releases one mg
of glucose from soluble starch in 1 hour at 30ºC.