Amylase Enzyme Assay

Aim:

To determine the amylase activity of given bacterial culture.

Introduction:

            Starch is an abundant carbon source in nature. α-amylase (1, 4 α D-glucanohydrolase; EC 3.2.1.1) hydrolyses α-1, 4-glucosidic linkage in starch and related molecules. It is one of several enzymes involved in starch degradation. Amylases constitute one of the most important groups of industrial enzymes being extensively used in food, textiles, paper, brewing and distilling industries. Most of the available amylases produced commercially are of microbial origin. The enzyme is widespread among aerobes and anaerobes. Gram positive bacteria, particularly the genera Bacillus and Clostridia are prolific producers of amylases.

Principle:

The α -amylase activity is measured using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. In this method, starch by α –amylase is converted into maltose. Maltose released from starch is measured by the reduction of 3,5-dinitrosalicylic acid.

                         

                        α –Amylase

Starch + H2O                            Maltose (reducing agent)

Maltose reduces the pale yellow coloured alkaline 3, 5-Dinitro salicylic acid (DNS) to the orange-red colored. The intensity of the color is proportional to the concentration of maltose present in the sample.

This intensity change in color is measured using a  spectrophotometer as the absorbance at 540nm wavelength. Wave length is set to 540 nm because it is the region where orange-red color absorbs.

Materials Required:

DNS Reagents:

 DNS reagent contained DNS (1%), potassium sodium tartarate (Rochelle salt, (1 M) and NaOH (0.4 M) and ddH2O. 3.6.1.1. Procedure for 100 mL Reagent Dissolve by stirring at room temperature (RT) 1g DNS in 50 mL ddH2O, then added 20 mL 2 M NaOH and 28.2 g Rochelle salt, finally made up to 100 mL by ddH2O. The reagent was stored at RT. 3.6.2. Other solutions

Buffer:

0.02 M sodium phosphate buffer (pH, 6.9) with 0.006 M sodium chloride.

Starch solution:

1.0% starch solution was prepared fresh by dissolving 1.0 g soluble starch in 100 mL 0.02 M sodium phosphate buffer (pH, 6.9).

Maltose stock solution:

Dissolve 100 mg maltose in 100 ml of dist. water.

Maltose Working Standard:

Add 10 ml of maltose stock solution in 100 ml of of dist. water.(Concentration 100 µg/ml)

Procedure:

Ø Pipette out 0.5 mL enzyme solution and incubate tubes at 25^oC for 3 min.

Ø Add 0.5 mL starch solution and incubate for 5 min (RT).

Ø Stop the reaction by adding 1 mL DNS reagent.

Ø Heat the solution in a boiling water bath for 5 min.

Ø Cool it in running tap water.
Ø Make up the volume to 10.0 mL by the addition of ddH2O.

Ø Read the absorbance at 540 nm using UV-Vis Spectrophotometer

Ø Blank is prepared without enzyme.

Ø Prepare a standard graph with 0 -100 μg maltose.

Result: