INTRODUCTION Starchy substances constitute the major part of the human diet for most of the people in the

world, as well as many other animals. They are synthesized naturally in a variety of plants. Some plant examples with high starch content are corn, potato, rice, sorghum, wheat, and cassava. It is no surprise that all of these are part of what we consume to derive carbohydrates. Similar to cellulose, starch molecules are glucose polymers linked together by the alpha-1,4 and alpha-1,6 glucosidic bonds, as opposed to the beta-1,4 glucosidic bonds for cellulose. In order to make use of the carbon and energy stored in starch, the human digestive system, with the help of the enzyme amylases, must first break down the polymer to smaller assimilable sugars, which is eventually converted to the individual basic glucose units Alpha-amylases are found in plants and in animals. Human saliva is rich in amylase, and the pancreas also secretes the enzyme. Individuals from populations with a high-starch diet tend to have more amylase genes than those with low-starch diets; chimpanzees have very few amylase genes. It is possible that turning to a high-starch diet was a significant event in human evolution. Beta-amylase cuts starch into maltose units. This process is important in the digestion of starch and is also used in brewing, where the amylase from the skin of the seed grains is responsible for converting starch to maltose (Malting, Mashing). This experiments explain the effect of subtrates concentration, pH and temperature on the enzyme activity. This can be done by using alpha- amylase to catalyzed the stacrh hydrolysis. The pH of the environment has a profound affect on enzyme activity and stability. Many enzymes are only active within a fairly narrow range of pH and exposure to pH values outside this range can lead to irreversible loss of activity. However, the optimal pH's for the biological catalysts produced by most commercial strains of microorganisms lies between pH 4.0 and 7.5. Since enzymes are made up at least partially of protein, they are sensitive in varying degrees to heat. Raising temperatures of the environment generally multiplies the degree of activity by the enzyme. Optimal temperatures generally range from 37 C to 60 C for most hydrolytic enzymes. High temperatures (over 66 C) generally have detrimental effects on the enzymes.

OBJECTIVE 1. To study the several parameters those affect the kinetics of alpha-amylase catalyzed hydrolysis of starch.

METHEDOLOGY

Assay of amylase A. Effect of Substrate Concentration

1. Determine the activity of amylase by mixing 1.25 ml of 1%, 3%, 5%, 7% and 10% soluble starch, 0.25 ml of 0.1M sodium acetate buffer (pH 5.0), 0.25 ml distilled water and 0.25 ml of crude enzyme extract in a test tube. (Add 357 ml 0.1M acetic acid and 643 ml 0.1M sodium acetate for preparing 0.1M sodium acetate buffer at pH 5.0)
2. Incubate the test tube containing the reaction mixture at 50oC for 10 minutes by using

water bath. 3. After 10 minutes incubation, estimate the liberated reducing sugar equivalent to glucose through calorimetric analysis, using DNS (3, 5 dinitrosalicylic acid) method.

B. Effect of Temperature

1. Repeat procedure A by using 1% of soluble starch.

2. Incubate the test tube containing the reaction mixture at 30oC, 40oC, 50oC, 60oC, and

70oC. 3. Continue the experiments as procedure above.

Effect of the pH

1. Repeat procedure A by using 1% soluble starch at different pH of sodium acetate buffer as presented in Table 1 below.

2. Continue the experiments as in procedure A.

ph 3 4 5 6

vol. of 0.1M acetic acid 982.3 mls 847.0 mls 357.0 mls 52.2 mls

vol. of 0.1M sodium acetate 17.7 mls 153.0 mls 643.0 mls 947.8 mls

Table 1: Buffer solution preparation at different pH.

DNS (3, 5 dinitrosalicylic acid)

1. Prepare DNS reagent by dissolving 5 g of DNS and 8 g of NaOH in 100 ml of distilled water. 2. Make up the volume of solution up to 300 ml. 3. Add potassium sodium tartarate slowly to the solution until all DNS dissolved and the volume once again make up to 500 ml.

4. Pipette 1.5 ml of sample into a test tube and add 1.5 ml of DNS reagent. Boil the reaction mixture for 5 minutes and allow to cool down before adding 10 ml of distilled water to the test tube. 5. Read the absorbance of the mixture at 575 nm by using spectroquant 400. 6. Prepare a standard curve of 1 g/l in range of 0-400 mg/l. A standard solution of range of 50, 100, 150, 200, 300 and 400 mg/l had been chosen to develop a standard curve.