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Introduction Starch is composed of ? -amylose and amylopectin. The structure of ? -amylose consists of long polymer chains of glucose units connected by an ? (1-4) linkage. Amylopectin consists mainly of ? (1-4) linked glucose residues but is a branched molecule with ? (1-6) branch points every 24 to 30 glucose residues on average. As a result of the bond angles in the ? (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. ?-amylases are found in saliva, pancreatic juice, human breast milk, serum and certain tissues such as the liver. This enzyme catalyzes the hydrolysis of ? (1-4) linkages in starch by breaking it down to maltose and some glucose. As the starch is broken down, the coiled structure of ? -amylase is unfolded. Therefore, iodine will no longer be able to form the blue complex with the ? 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 Amylose Starch coiled at ? (1-4) linkage Amylopectin 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 ? -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 Objective The aim of this experiment is to determine the effect of temperature and pH on the enzyme activity of ? -amylase . In this experiment, the reaction of ? amylase with starch will be carried out at different temperatures and pH values with all other conditions constant. Method Refer to lab manual. Results 1. The Effect of Temperature on Enzyme Activity Tube No. 0. 1% Buffered Starch (ml)Water (ml)0. 0044% ? -Amylase (ml) Enzyme A0. 0044% ? -Amylase (ml) Enzyme BWaterbath Temp (??C)A680Amount of product formed (P) 15-1-350. 1080. 116 25–1350. 1510. 073 35-1-500. 1130. 111 45–1500. 1420. 082 55-1-700. 240-0. 016 65–1700. 681-0. 457 75-1-600. 258-0. 034 85-1-800. 2120. 012 951–350. 156-control 1051–350. 92-control 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% ? -Amylase (ml)A680Amount of product formed (P) 1561-0. 015-control 2561-0. 008-control 353-10. 013-0. 0015 454-10. 0110. 005 555-10. 018-0. 0065 656-10. 018-0. 0065 757-10. 0110. 0005 858-10. 012-0. 0005 959-10. 019-0. 0075 10510-10. 024-0. 0125 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 ? -amylase Activity Fig. The Effect of pH on ? -amylase Activity Discussion The Effect of Temperature on Enzyme Activity Enzymes are protein in nature.
The activity of enzyme and the rate of product formed will be maximum at the optimum temperature as temperature affects the speed of the molecules. Once the temperature increases above the optimum, the enzyme denatures. Denaturation is the loss in the shape of the active site of the enzyme. Therefore, the substrate cannot bind to the active site and at the same time the enzyme-substrate complex cannot be formed and the reaction cannot proceed. According to the results from the temperature graph, it can be assumed that the optimum temperature for enzyme A and B is 35 ‘C and 50’C respectively.
This is due to the fact that the lower the absorbance value, the greater the enzyme activity and the greater the amount of product formed. The graph showed that the activity of enzyme was the highest at 35’C and 50’C and the highest amount of product was formed as the most amount of starch is broken down. The control solutions were carried out to determine the amount of product formed as it indicates the total amount of starch present before the enzyme has hydrolyzed the starch. The Effect of pH on Enzyme Activity Enzymes are also adapted to operate at a specific pH or pH range.
The pH corresponding to the highest rate of reaction is the optimum pH. At extreme pH, low or high, enzyme becomes denatured and loses the activity as the shape of the enzyme is changed by the denaturation. It can be assumed that at low pH, where hydrogen ions are in abundance, enzyme becomes protonated and loses its negative charge and at high pH, with abundance proton acceptors, enzyme ionizes and loses its positive charge. Therefore, the electrical attraction between polar groups in the enzyme will be altered. By changing the pH, the enzyme activity can be corrected easily.
Due to the results obtained from the pH graph, it can be determined that the optimum pH for the enzyme (? -amylase) is either pH 4 or 7 . Similar to the temperature data interpretation, the enzyme activity was the highest at both pH 4 and 7 , and the amount of product produced was also the highest. In this experiment, the control solutions were also carried out to determine the amount of product formed. The absorbance values at pH 8,9 and 10 show minus sign, so the values at these points were considered as outliers because of negative product formed.
The experimentally derived answers were different from the results of previous experiment which has shown that the optimum temperature for enzyme A was 70’C, 80’C for enzyme B, and the optimum pH for ? -amylase was 7. This could be due to many factors. The possible reasons are: -The starch-buffer mixture was not sufficiently mixed because starch is in suspension and it need to mix thoroughly before using every time for the reaction. -The turbidity of the solution in cuvette can alter the absorbance readings. -The ? -amylase enzyme was left in the test tube for too long. The water bath temperature was not maintained properly. Conclusion According to the results obtained from the experiment, there were many minus signs for the amount of product formed. It means that more starch were present in the reaction test tube than the original starch solution. This result was wrong and there could be turbidity or cloudiness of solutions. This can lead to great error in absorbance measurements. Human error in handling the samples and apparatus could also be the factors for getting false result. Moreover, maximum activity of enzyme varies with the incubation time.