Found in the saliva and pancreatic secretions of animals including human beings as well as the plant seeds, bacteria and fungi (Squid et al. , 2010), the enzyme a-amylase that was studied during the experiment has significant impact on the hydrolysis of starch. By breaking the alpha, 1-4 glycoside linkages in the carbohydrates, amylase hydrolysis the starch, a polysaccharides that is stored in plants and cannot be directly digested by animal cells, into maltose, a disaccharide that later generate two units of glucose to undergo metabolisms and provides necessary energy (Slaughter et al. , 2001 ).
The enzymatic activity of a-amylase is facilitated by calcium and chloride ions during the hydrolysis (Marina, 2006 and Squid et al. , 2010). The complete digestion of starch and formation of maltose and glucose can be examined through the iodine test when ASK’ reagent is added into the solution and remains brown instead of turning into dark blue, marking that all the molecules of starch have been fully hydrolysis (Hands, 1932). While amylase effectively activates the hydrolysis of starch, the efficiency of the catalytic process is influenced by several factors including temperature, pH level and the concentration of the substrates etc.
In this experiment, as the a-amylase s a type of protein, the efficiency of enzyme is highly related to its hydrogen bonds which are affected by the temperature. Though the enzyme is collected from the porcine pancreas, due to its structural similarities to amylase in human bodies, the behaviors of two amylase should resemble each other. Given that under extreme temperature enzymes will be denatured and unable to function and the constant temperature of pigs is around ICC, the hypothesis of this experiment is that at 37 DC amylase will catalyst the hydrolysis with the highest speed, followed by amylase at ICC.
Amylase at CO will react extremely slowly u to the crystallization of hydrogen bonds and attacks, amylase will lose its function since it will be denatured. Materials and Methods Four test tubes were marked from AAA to AAA. Then, ml of 1% starch solution from Carolina Biological Supply Company, ml of denizen water and 1 ml of 6. 8 hydration buffer from IVR International/ Micro Essential Laboratories were added into each tube. Another four test tubes were also labeled from Bal to BE and added ml of 1% a-amylase from porcine pancreas from Sigma Aldrich. Eight tubes were paired according to the same number (Land Bal etc. And assigned to environments at different temperature: Tube AAA and Bal were placed into a water bath at ICC; Tube AAA and BE were placed into a water bath at ICC; Tube AAA and Brewer placed on the tube rack (at about ICC); Tube AAA and BE were placed into an ice bath at CO. All test tubes were kept at different temperatures for 10 minutes. Meanwhile, a control group of starch solution was prepared without amylase. (Bio Lab Manual, 201 3) At the same time, a test plate was added 2 drops of kill reagent (1% Iodine and 2% SKI) from Carolina Biological Supply Company per well.
After 10 minutes, when est.. Tubes were still in the original environments, solutions in Tube AAA with Bal were mixed and a timer was started. At each 30-second-interval, a drop of the mixture was released into the well on the test plate until the solution in the plate did not change into dark blue and remained brown, indicating the end of the reaction by showing no presence of starch and presence of maltose and glucose. The experiment was repeated on the tubes at other temperatures. Slow reactions were observed and recorded up to 420 seconds due to time limit.
Data were pooled from each bench and average and standard deviation were calculated. The data of the control group were also obtained. Results Figure 1 The test plate of iodine test under different temperature. Dark blue wells indicated the presence of starch while the brown ones indicate the completion of starch hydrolysis. (Upper half: 37 DC; Bottom Half: CO) The rate of reaction was fastest at 37 co (n=4, mean=212. As, SD-66. 1 s) while the rate of reaction at ICC was only slightly less than it (n=4, mean=217. As, SD-61. As).
On one hand, at 37 co the amylase showed the greatest efficiency in catalyst the hydrolysis of starch. At the same time, the amylase also showed considerable catalytic efficiency at ICC. But on the other hand, when temperature dropped or rose to extreme value such as CO or 1 COO, the function of amylase was inhibited and such biochemical transformation of substances could hardly process. This result obtained is consistent with the reality that during normal body temperature, regardless of pig or human beings, amylase is able to catalyst the hydrolysis of starch with the highest speed.
Therefore, we may conclude that even taken out from where it was found, the amylase still maintain its original biochemical repertories. The experiment did not show the biochemical mechanism of the modification from temperature to amylase activity. However, according to the scientific research done by other scientists, a temperature that ranges from 20-ICC could make structures including weak interactions, hydrogen bonds and disulfide bridge exist within and stabilize the enzyme molecules to maximize their activities.
At the water freezing point (CO), the hydrogen bonds are crystallized and become more constrained and less flexible while at high temperature like ICC, the bonds consume certain energy to become unstable and fragile, either of which contribute to the proper functions of amylase (Atomic et al. , 2003). While the result of the experiment perfectly matched what was expected, however, such conclusion could only be made at qualitative phase and it is obvious that weakness of this experiment existed and prevented the further understanding of amylase at quantitative level.
Several modifications to the current experimental designs could be made to enhance its accuracy. Firstly, the sample size needs to be expanded. With only four groups, the data was so limited. As a result, the data had great standard deviations of more than 60 seconds. Simultaneously, the random errors were at high possibility to take place. Therefore, with the increase of sample size, the data can be more accurate and stabilized and potential random errors could be discarded to ensure the coherence of the data.
Furthermore, even though neither the test tube at CO and ICC enabled the completions of starch hydrolysis, the reasons of the two groups are not the same. Therefore, in order to detect the reason of the loss of catalytic ability, follow-up experiments need to be practiced. A possible design might be to change the test tubes from CO or ICC into ICC for another 10 minutes then redo the iodine test to see this time whether the amylase can function well or not.
This manipulation will convince the hypothesis about the reason behind the superficial phenomena that was shown in the original experiments and present the difference between denaturing of protein and crystallization of hydrogen bonds. It is important for people to thoroughly understand the amylase activity and all the factors that are potentially capable of influencing such activity through which people can understand how human bodies work as well as the physiology of other organisms. At the same time, the research in amylase activity could potentially bring economical benefits to industrialized starch products manufacturing.