Temperature vs. Enzyme

Enzymes are proteins that help with digestion and metabolism. Their function is to speed up certain reactions within cells. In this case, enzymes were part of a reaction with iodine. A total of eight test tubes were used; four for bacterial amylase and the other four for fungal amylase. A spot place was used to see the reaction between the amylase and iodine. Four out of the eight test tubes were labeled with the letter ‘S’ for starch solution. Each test tube had a designated temperature: zero, twenty-five, fifty-five, eighty-five and they were all in degree Celsius. They were placed in water baths for five minutes. They had a time of two minutes so that two to three drops of either bacterial or fungal amylase were placed into the well with the two drops of iodine. The rest of the experiment was repeated another four intervals. As for the results, it was observed that as the temperature increased, the more starch was present within the reaction of the amylase. For example, in the eighty-five-degree Celsius column/four-minute row, the color in the well was a dark purple, which means iodine had reacted with the amylase. Since the bacterial and fungal were used to their environmental, or optimal temperature it was expected to react with the iodine in the hotter temperature. During lab time, it was discussed how the results would come out and it ended up supporting the hypothesis.

Introduction

Enzymes have been existing since the 1800’s and ever since then, they have been very important for experiments and in the human body. Enzymes are biological catalysts that speed up reactions and lower down the energy within cells. Without enzymes, nothing would be able to break down or properly work. There are factors that can affect the way the enzymes work such as temperature, pH levels and concentration of certain substances. Temperature affects the enzymes the most because of its optimal temperature. Enzymes have their own optimal temperatures because of their environment living conditions. If at any point their optimal temperature gets higher, their active site changes its shape and it does not allow it to bind to their substrates. It can also relate to enzyme activity. Enzymes can work with starch to see the reaction between them. Starch is a carbohydrate that breaks down into glucose and used as energy after digestion. In this experiment, the class worked with enzymes and iodine. There were two types of enzymes that were used which were the fungal amylase, Aspergillus Oryzae, and bacterial amylase, Bacillus Lichenformis. The class used these enzymes to identify how they are affected by different types of temperatures and the concentration levels in the Celsius range and how the starch had the ability to break down into maltose. Anyone can tell if starch is present in a reaction by examining the color in turns to. If starch is present, the yellow color will turn into a blue-black color. If starch is not present in the reaction, it will stay in the yellow color. Bacterial amylase’s optimal temperature is at a high temperature because of their stability to heat. Fungal amylase is acidic on the pH range and it also does not have an optimal temperature as high as the bacterial amylase does. Fungal and bacterial amylase have similar optimal temperature (over thirty-two degree Celsius or eighty-five degrees Fahrenheit), because they are already used to it due to their environmental temperatures. If they are placed in an environment where their optimal temperature is changed, they die.

There are many enzymes that speed up chemical reactions in our bodies. They also work to break down food while it is digesting, communicate with other cells, keeps cells alive and does much more. If it were to not work properly, it would work very slow and will not be able to keep the body going. Enzymes are necessary for our bodies and to constantly be regulating. The activity of the fungal and amylase will increase as the temperature increases.

Methods

To begin the lab, two spot plates were set up on top of napkins with the “Temperature (in degrees Celsius” written across and “Time (in minutes)” written down the side. 0 degrees, 25 degrees, 55 degrees and 85 degrees were the temperatures. 0, 2, 4, 6, 8 and 10 were the times recorded. Four test tubes were placed in the table and labeled with its designated temperature, bacterial/fungal amylase and the group number. Another set of four test tubes were placed on the table and labeled with its designated temperature, bacterial amylase/fungal amylase, group number and the letter ‘S’ (starch solution). A milliliter of 1.5% of starch solution was added into the test tubes that were labeled ‘S’. After that, one milliliter of amylase was added into the test tubes that did not have any starch solution. All four tests tubes that had starch solution and the four test tubes that had amylase were placed into their designated temperatures. The zero degree Celsius was placed into the ice bath, the twenty-five degree Celsius was placed into the twenty-five-degree water bath, the fifty-five degree Celsius was placed into the fifty-five-degree water bath and the eighty-five degree Celsius was placed into the eighty-five-degree water bath.

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All four tubes were placed into their designated temperature baths for five minutes. During those five minutes, two to three drops of iodine was added to each well in the zero minutes row. After the five minutes were up, a few drops of starch solution from each of the temperatures were transferred onto the spot plate on the zero minutes row. This was done without removing the tubes from the water baths. Small pipettes were used to transfer the solutions. Each of them were labeled with their appropriate temperature to be reused for each treatment and to not get mixed-up results. In each interval, the starch solution was poured into the test tubes that had amylase. The timer was set for two minutes as soon as amylase was added. After the two minutes were up, the pipettes were used to suck in a few drops off the starch-amylase solution from each of the test tubes. Two to three drops of it were placed in the second row of the spot plate. The color change and observations were recorded in the both data tables. For the rest of the experiment, two minutes were added, and the previous two steps were repeated three more times. After ten minutes went by, any observations were noted. The experiment was repeated using the other amylase.

Discussion

Overall, the results did support my hypothesis. In the results, it showed that as every two minutes, while the temperature increased, the hydrolysis ratings increased, which means that the ratings of four and five, there was starch present when iodine was dropped into the wells. On the scale from one through five, five means that starch is present, and the color turned from yellow to a dark color, like blue or black and one means that there was no fungal or bacterial activity and starch was not present. When the color of the solution once iodine was added is yellow, there was no activity occurring because starch was not present.

For the bacterial amylase results, in the zero minutes row, it went from a light green color to a dark blue color as the temperature increased. In the two minutes row, it went from a brown color to a dark purple color as the temperature increased. The same results repeated for the rest of the experiment. It was determined that starch had been catabolized because of the distilled water and the different temperatures (Alberte et al. 2012). The independent variable was the temperature of the water which was zero degree Celsius, twenty-five degree Celsius, fifty-five degree Celsius and eighty-five degree Celsius. The dependent variables were the number of drops of iodine, which were two to three drops into each well before the bacterial or fungal amylase were dropped and the time intervals, which were at zero minutes, two minutes, four minutes, eight minutes then ten minutes. The only control variable in this experiment was the type of enzyme. Starch should not be present at zero degrees because the water was very cold and no activity occurred, but at eighty-five degrees Celsius, starch was reacting with the hot water and the iodine. The fastest rate of reaction for fungal and bacterial amylase was at eighty-five degrees because of the hot temperature and the intensity of the iodine reacting with the amylase. The fungal and bacterial amylases are used to hot temperature. There could have been some source errors for this lab. For example, the water bath could have been too hot than what it said in the lab manual.

There could have been a cross contamination with another substance, which can cause a mess up in the results. At first, it was believed that fungal and bacterial amylase would have an increase in enzyme activity because of the temperature. That hypothesis stayed throughout the entire experiment and the results that were recorded were as expected because we had learned that the hotter the temperature, the more activity there will be between the amylases and the iodine, and the nature of the enzymes. There are some limitations with the materials for this experiment. The given times in the procedure were important because if the test tubes were to be in the water bath for over five minutes, the amylase can denature or get to hot and throw off the results. The amounts of drops of iodine or the bacterial and fungal amylase are supposed to be between two to three drops because of the concentration and enzyme activity between the iodine and the amylases.