Enzymology

Experiment 3

Aim of the Experiment

Enzyme estimation, activity and other parameter.

Principle:

Amylase assay:

Amylase assay was measured by following the methodology proposed by Miller (1959). A reaction mixture containing 0.5ml of 1% soluble starch solution prepared in 0.1M phosphate buffer (pH 6.5) and 0.5ml of (crude enzyme) culture supernatant. Appropriate controls were also maintained. The mixture was incubated for 30 min at room temperature. After incubation, the reaction was terminated by adding 1ml of Dinitrosalycilic acid (DNS) solution (1g of DNS dissolved in 20 ml of 2M NaOH, to which 30g of sodium potassium tartarate was added and filled with distilled water to 100ml). After termination, the reaction mixtures were boiled for 5 min to develop brown color. The final volume was made to 5ml with distilled water. Absorbance was measured at 540 nm. One unit of enzyme activity was defined as the amount of enzyme that releases 1µ mol of reducing sugar (glucose) in 1min under the assay conditions. The enzyme activity was calculated using the following formula:

Amylase present in the supernatant (U/ml) = Micromoles produced/ Volume of the sample X Incubation time of the sample.

Determination of α-amylase activity

History:

• α-Amylases (EC 3.2.1.1) is an enzyme of glycoside hydrolases mainly produced in the salivary glands and pancreas, play a well-known role in hydrolyzing a-1,4- glucosidic bonds between glucose in starch ( consists of two types of polysaccharide amylose, amylopectin) and maltose is release. Elevated level of α-Amylases in serum can be used as markers for clinical diagnosis of diseases, e.g. Pancreatitis. When the pancreas is diseased or inflamed, amylase releases into the blood.

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.

Starch + H2O α-Amylase 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.

This procedure applies to all products that have a specification for α-amylase.

Materials required

1. 0.02 M Sodium phosphate buffer ph-7

2. 1% Starch

3. 2 N Sodium hydroxide

4. Sodium potassium tartrate tetrahydrate

5. Dinitrosalicylic acid color reagent

6. Standard Maltose Stock Solution 1mg/ml

7. Amylase enzyme

Procedure

Adjust spectrophotometer at 540 nm and 25°C.

Calculation

Enzyme activity = OD (test) x concentration of St (µmoles) x dilution of enzyme

OD (st) x incubation time (3 min)

= µmoles maltose formed / min/ml.

Observation & Result:

Mark the O.D and Then plot the graph for the same.

Reference Material

1. Enzymes- by Palmer.