Amylase Activity in Different Environmental Factor

The Effects of Temperature, Concentration and pH on the Enzymatic Activity of

Introduction

Enzymes are globular proteins that act as biological catalysts for biochemical processes within a living cell. Enzymes increase reaction rate by lowering the activation energy of the reaction, however they are not consumed. An enzyme is present as both a reactant and a product in a reaction. Substrates are the reactants for an enzyme and since enzymes have such a high specificity, only specific substrates will fit on certain enzymes. These substrates bind to the active site of an enzyme and form enzyme-substrate complexes. Weak interactions such as hydrogen bonds and ionic bonds hold the substrate in the active site, while the substrate is transformed into product by side chains of amino acids. The enzyme then releases the newly formed product, remaining unchanged and ready to catalyze another reaction (Campbell, Reece 2008). In this experiment we will use fungal amylase to determine how the enzymes function in different environmental factores.

Amylase, an enzyme that is found in saliva and fungi , catalyzes the hydrolysis of starch (amylase). Since enzymes are proteins, their secondary and tertiary structures are affected by temperature, pH, and the different concentration of the amylase . Enzyme activity is closely associated with the structure of an enzyme, so any change in the secondary or tertiary structure leads to a change in enzyme activity. In this experiment we examined the enzymatic concentration , the effect of temperature and pH on the activity of fungal amylase . Molecular iodine forms a complex with starch that has a characteristic deep blue color. As starch undergoes hydrolysis to form oligosaccharides and glucose, the characteristic of color of the starch-iodine complex disappears or turns to yellowish-brown.

Therefore, loss of the deep blue color can be used to measure of the extent of hydrolysis of starch.

Amylase turns starch polymers into monomers such as maltose. Therefore, if amylase has done its job right, there should be no presence of starch when the amylase is at If temperatures are too cold, the molecules themselves move slower and take much longer to react until they finally cease moving all together if temperatures are low enough. Temperatures that are too high, on the other hand, cause the enzyme to denature and thus not allow the reactions to take place between enzyme and molecule because they no longer fit together (Goldina and Simms, 2010). In this experiment it is hypothesized that the amylase/starch solutions will not hydrolyze at 100% in either the ice water bath (-4?) nor in the 80? bath. This is hypothesized because human and fungal amylase is being tested, and neither human nor fungus' thrive in extreme temperatures..

Methods

The first experiment we will examine the effect of enzymatic concentration on amylase. Four standard test tubes were labeled D1-D4, filled with 1 ml of amylase soulution, three test tubes had 3ml of pH7 Buffer. One tube was placed in 4C, second tube was placed in 22C and the third tube was in 50C water. They were allowed 12 minutes to reach the respective temperatures. Next, we had three tubes with 1-2 droppers of potato juice, which were placed in different temperatures thesame way as buffer tubes. Then, we had tubes with 1-2 droppers of catechol placed in different temperatures the same way for 12 minutes. After 12 minutes, we added catechol and potato juice to the buffer tube accordingly to the same temperatures. The same procedure was repeated 7 times with the intervals of 2 minutes between each measurement of the tube with the respective temperature. The results were recorded in terms of absorbance reading at 420nm. The Effects of pH on Enzyme activity: five test tubes 3mls of pH 4, pH 6, pH 7, pH8, or pH 10 added. Then each test tube had 350mls of potato juice added. After adding potato juice, 350mls of Catechol was added to each test tube and then covered with Para film. After being covered, the tubes were inverted several times to mix the buffer and pH. Finally the tubes were allowed to stand for 5. The results were recorded as absorbance readings at 420nm.The Effects of Enzyme Concentration on Enzyme Activity: four test tubes were taken and had 3mls of pH 7 phosphate buffer added to them. Test tube 1 was given 700ul of additional buffer. Test tube 2 was given 525ul of additional buffer. Test tube 3 was given 350ul of additional buffer. Test tube 4 was left only with 3 ml of phosphate buffer. Then none of potato juice was added to test tube 1. 175ul of potato juice was added to test tube 2. 350ul of potato juice was added to test tube 3 and 700ul was added