How Does Temperature Affect Bacterial Amylase

Abstract
This experiment analyzes the effects of how temperature affects bacterial and fungus amylase and also discovers the optimum temperature for these enzymes. The amylase was mixed with starch at temperatures of 0℃, 37℃, 57℃, and 90℃. Iodine was added to each mixture and colour changes in each case. Bacteria amylase was found to be effective at 55 0C as the temperature dropped drastically from 4.58℃ to 2.33℃. This shows that the amylase catabolized a lot of starch hence little is left which cannot turn the iodine solution to blue-black which indicates the presence of starch. Fungal amylase is denatured at 90 ℃. This was shown by the rows remaining blue-black which indicate the present of plenty of starch. It was expected that amylase
catabolized the starch; the colour of iodine should become lighter and lighter as the data drops. However, this is not the case, the data fluctuated and sometimes increased instead of decreased. The optimum operating temperature of bacterial amylase is lower than 550C while that of fungal amylase is lower than 90 ℃.

Introduction
Enzymes are biological molecules in charge of many chemical reactions that are needed to sustain life.
It is protein in nature and biological catalyst, which speeds up the reaction. Enzymes speed up the reaction by lowering the activation energy (Keener, 1998). Catalyst is a substance which speeds up the rate of reaction by lowering the activation energy but do not take part in the reaction (Lerner, 1988). Enzymes are specific in nature and they catalyze specific reaction. Active site in the enzyme is where it binds with the surface of the substrate to form enzyme-substrate complex. The binding of the enzyme to the substrate can either be lock-and-key model in which the enzyme perfectly fits and binds to the substrate (Segel, 1975). After binding to the active site, the rate of reaction is increased by the enzyme by changing its shape. Another model is an induced fit model where the binding energy is used to distort ways substrate bond to the enzyme. We expected the colour of iodine to turn blue-black when it mixes with iodine and because the amylase is breaking down the starch, the iodine presence will react with few starch remaining hence the colour would not turn to
blue-black.
Different enzymes have different optimal conditions needed to catalyze the specific reaction. This activity can be affected by factors such as pH, temperature and presence of inhibitors (Cossins, 2012).
In this experiment, we studied the effects of temperature on enzymes found on living organisms that break down starch to determine the fungal and bacterial amylase optimal temperature.
The type of the enzyme tasted in this experiment was amylase. Amylase enzymes break down starch into small carbohydrate molecules called monomers, or saccharides if it is sugar. Starch is broken down by amylase into maltose or glucose and fructose, which are simple sugars (Guzmán‐Maldonado, 1995).
There are two types of amylase that are being tested in this experiment, which are Aspergillus oryzae from fungus, and bacteria. Bacteria and fungi are completely different organisms because bacteria are prokaryotes while fungi are eukaryotes. The optimum conditions of amylase on both are expected to vary because of this difference. Fungi works in the optimum temperature of 57℃ while bacteria thrive in extreme high temperatures. Bacteria thrive in this temperature because it cannot breakdown starch at a lower temperature (Jay, 2012).
Enzymes work best in a small range of temperature mostly in optimum temperature. At a lower temperature, the reaction is slower because the hydrogen bonds and the hydrophobic interactions are not fast enough to allow to induce fit (Echlin, 2013). The enzymes maintain a strong shape depending on the strength of hydrogen bonds. Weak bonds allow minimal range of conditions while wide range allows optimal conditions to occur (Hochachka,
1973).
We expected the colour of iodine to turn blue-black when it mixes with iodine and because the amylase is breaking down the starch, the iodine presence will react with few starch remaining hence the colour would not turn to blue-black.

Methods
Sixteen test tubes were split into eight and eight. One set of eight was moved to one side and we focused on the other eight. The group focused on eight test tubes then further split into two groups of four test tubes. Blue and pink tape was used to distinguish tubes. All eight test tubes were labelled with a letter "F" for Fungal Amylase. Different degrees were labelled on the other four test tubes, 0 0C, 37, 570C, and 900C; also one of these test tubes had a letter 'S' for starch. Four test tubes had "F (temperature) and the other four were labelled "FS (temperature). Five ML of 1.5% of starch were added into the test tubes that only had and ‘S’ marked on them. One ML of Fungal Amylase concentration was added to the other 4 test tubes. Eight test tubes were placed in ice water and 3 drops of iodine were added to each spot plate and allowed to incubate for 5 minutes. 3 drops of starch solution were pipetted and placed in a tile rows grouped by their temperature. Amylase solution was added and starch liquid was added into amylase solution at their own temperature. The colour reading of each tile was recorded after 10 minutes. The same process was repeated with bacterial amylase.