The Effect of Substrate Concentration on the Enzyme Amylase

LAB 10:
NAME: DaeNia La Rodé
DATE: 25TH January, 2011.
FORM CLASS: L6 3
SUBJECT: Biology
TITLE: Enzymes
AIM: To investigate the effect of substrate concentration on the enzyme amylase
INTRODUCTION:
Enzymes are perhaps one of the most important proteins of the human body. Enzymes such as amylase, an enzyme that breaks down carbohydrates, work by means of surface catalysis. In other words, the surface of the enzyme enables other molecules to react in a manner they would not be able to without the surface of the enzyme present. Enzymes achieve this by lowering the amount of activation energy needed for anabolic reactions, allowing these reactions to occur as catabolic reactions would. Chemical reactions require an amount of energy to start them off; this is known as activation energy. Enzymes are generally large proteins made up of several hundred amino acids, and often contain a non-proteinaceous group called the prosthetic group that is important in the actual catalysis. In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to the active site of the enzyme. The enzyme and substrate are held together in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds, and ionic bonds. Enzymes act as catalysts to lower the required activation energy. Enzymes do this by weakening the covalent bond with a substrate molecule or by holding the substrate in a particular position that increases the probability of a reaction occurring between the molecules. Products can only be formed when effective collisions occur, this means that reactant molecules colliding is not enough to form effective collisions. Reactions can only occur when particles first collide. The collision present must have enough energy impact to overcome the Activation Energy; they must also have a proper positioning to be effective. Increase the concentration of the substrate and there will be increasing substrate molecules as compared to enzyme in the mixture. The more starch molecules there are, the higher the probability of collisions occurring however when the all the enzymes are occupied it said to have reached v-max. Consequently results in an increase in the number of effective collision, this means the rate of the reaction will increase until v-max.

Enzymes are not only important because they keep the metabolic pathways free of congestion but they are also important in, digestion. Enzymes are needed to perform an infinite number of tasks within the human cellular system. Without enzymes the body could not possibly function properly, let alone sustain life. Enzyme productivity like many other things in our body tend to vary based on outside factors. Environmental parameters such as temperature, pH and substrate concentration, such as starch, all cause changes in enzyme productivity.
The enzyme used in this lab was amylase, which is commonly found in saliva and germinating seeds. Amylase’s substrate is starch, a polysaccharide breaking it down into maltose, a disaccharide. Starch is one of the most common stores of carbohydrate. Starch is composed of a combination of two polymers; a linear polysaccharide- amylose and a branched polysaccharide, amylopectin. It is a polymer, which consists of a chain of amino acids linked together by glucose bonds and several double units. When amylase reacts with starch, it cuts off the disaccharide maltose (two glucose molecules linked together). As the reaction progresses, less starch will be present and more sugar (maltose) will be present. The activity of amylase was observed by using iodine. Iodine reacts with starch to form a blue black color. As amylase breaks down starch, less and less starch will be present and the color of the solution (if iodine is added) will become lighter and lighter. The color change was observed using spotting tiles.
MATERIALS/APPRATUS:
MATERIALS: amylase, starch suspension of six different concentrations (0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%), Water
APPARATUS: five (5) test tubes, spotting tile, five (5) Beakers, Forces, Glass Rod, Measuring Cylinder, Syringe, Dropper
PROCEDURE:
Five centimeters cubed of each starch suspension was placed into five labeled test tubes 1% of amylase was added to the test tubes and left for two minutes to allow for a reaction. One drop of iodine was added to each spot on a spotting tile. Two drops of the starch suspension and 1% amylase was added to each spot. This was done with six different percentages of starch; 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%. These individual percentages were done three times for accuracy.

RESULTS:
TABLE10.0: TABLE SHOWING THE TIME IT TOOK FOR THE IODINE TO TURN FROM A BLUE BLACK COLOUR TO BROWN
STARCH (%) TIME (seconds) CALCULATIONS AVERAGE TIME 1 2 3
1.0% 1 1 1 (1+1+1)/3=1 1
0.9% 1 3 2 (1+3+2)/3=2 2
0.8% 5 4 3 (3+3+3)/3=3 3
0.7% 5 5 5 (5+5+5)/3=5 5
0.6% 53 45 54 (53+45+54)/3=51 51
0.5% 97 97 97 (97+97+97)/3=97 97

TABLE10.1: TABLE SHOWING THE CALCULATED RATE BASED ON TABLE10.0

STARCH (%) CALCULATIONS (R=1/T) RATE
1.0% 1/1=1 1
0.9% 1/2=0.5 0.5
0.8% 1/3=0.33 0.33
0.7% 1/5=0.2 0.2
0.6% 1/51=0.02 0.02
0.5% 1/97=0.01 0.01

GRAPH 10.0: GRAPH SHOWING THE SUBSTRATE CONCENTRATIONS OF SUCROSE ON THE RATE OF REACTION OF AMYLASE

DISCUSSION:
If the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity. This is represented graphically in GRAPH10.0.
Outside experimentation, the body perhaps is the greatest test of enzyme adaptability. It uses enzymes in countless ways, and in countless conditions thus the importance of understanding the effects of its environmental parameters. Upon completion of the experiment it was found that when temperature and pH are held constant the activity of an enzyme system is determined by the relative concentration of the enzyme and its substrate, starch in this case. Specifically, we found 1% starch solution to result in the highest rate of enzyme activity. If there is an excess of substrate, the rate of catalysis is directly proportional to the enzyme concentration. If enzyme concentration is kept constant, as it was in the following experiment, then the rate of reaction is directly proportional to the amount of substrate present. Thus, an increase in substrate, starch, causes an increase in enzyme catalysis. However, this is only up unto the point when all enzyme molecules are utilized, or saturated. At this point the enzymes have reached their optimal catalyzing rate and will no longer increase in rate of productivity. A much larger experimental group would have been needed in order to find amylase’s optimal catalysis condition, or saturation point (V-Max).
There were many sources of error in this experiment such as the time delay posed by the fact that time was not properly compensated when all parts of the solution could not be added by one person simultaneously, or even the mixtures of the amylase and starch solutions could have been slightly off . All of these errors could have been reduced however through more careful planning and accuracy during preparation. In issues where reaction time was an issue, multiple people can add and remove samples. A limitation to this experiment was that only six different percentages of sucrose were used thus it could not be seen exactly where the quantity reaches its maximum amount of collisions between enzymes and substrate.
CONCLUSION:
It can be concluded that the greater the starch concentration the more collisions between the enzyme amylase and the substrate starch, however due to a lack of variation within the substrate concentration it cannot be determined at which point the amylase achieves v-max.