Starch is composed of α-amylose and amylopectin. The structure of α-amylose consists of long polymer chains of glucose units connected by an α (1-4) linkage. Amylopectin consists mainly of α (1-4) linked glucose residues but is a branched molecule with α (1-6) branch points every 24 to 30 glucose residues on average. As a result of the bond angles in the α (1-4) linkage, amylose actually forms a spiral much like a coiled spring. Amylose is responsible for the formation of a deep blue color in the presence of iodine. The iodine molecule slips inside of the amylose coil.The amount of blue complex that starch gives with iodine can be measured by using a spectrophotometer.
α-amylases are found in saliva, pancreatic juice, human breast milk, serum and certain tissues such as the liver. This enzyme catalyzes the hydrolysis of α (1-4) linkages in starch by breaking it down to maltose and some glucose. As the starch is broken down, the coiled structure of α-amylase is unfolded. Therefore, iodine will no longer be able to form the blue complex with the α-amylase. It can be assumed that the decrease in color (absorbance) is proportional to the product formed. In this experiment, the absorbance of the starch-iodine complex wil be measured at 680 nm in 1 cm cuvettes using single beam spectrophotometers. The intensity of the emerging beam will be decreased because the solution absorbs some of the radiation.
Amylose Starch coiled at α (1-4) linkage
The absorbance value(x) read from cuvette containing starch and water represents the total amount of starch-iodine complex. The absorbance value(y) read from cuvette containing starch, water and α-amylase at the respective temperature or pH represents the amount of starch-iodine complex which is left after the enzyme has hydrolyzed the starch.In order to get the amount of product;P (maltose and glucose) formed, need to subtract the (y) value from the (x) value. The amount of product formed (P) = x - y
The aim of this experiment is to determine the effect of temperature and pH on the enzyme activity of α-amylase . In this experiment, the reaction of α-amylase with starch will be carried out at different temperatures and pH values with all other conditions constant. Method
Refer to lab manual.
1.The Effect of Temperature on Enzyme Activity
Tube No.0.1% Buffered Starch (ml)Water
(ml)0.0044% α-Amylase (ml) Enzyme A0.0044% α-Amylase (ml) Enzyme BWaterbath Temp (°C)A680Amount of product formed (P) 15-1-350.1080.116
The average absorbance value of tube 9 and 10 minus the absorbance value obtained from each individual test tube is equal to the amount to product formed (P).
Product formed (P) = x-y
Average of control (x) (tube no. 9 and 10) = (0.156+0.292)/2 =0.224 Values of (y) = 0.108, 0.151, 0.113, 0.142, 0.240, 0.681, 0.258, 0.212
2.The Effect of pH on Enzyme Activity
Tube No.0.1% Starch (ml)1ml Buffer
pHWater (ml)0.01% α-Amylase (ml)A680Amount of product formed (P) 1561-0.015-control
The average absorbance value of test tube 1 and 2 minus the absorbance obtained from each individual test tube is equal to the amount of product formed (P).
Product formed (P) = x-y
Average of control (x) (tube no.1 and 2) = (0.015+0.008)/2 = 0.0115
Fig. The Effect of Temperature on α-amylase Activity
Fig. The Effect of pH on α-amylase Activity
The Effect of Temperature on Enzyme...