Amylase Production In Bacillus Subtilis Lab Report

DeAngelo, Eric, fundamentals of biology laboratory manual, LCCC,2013, Chapter 5 Cell The Effects Environmental Temperature and pH have on the Activity of Porcine Pancreatic Amylase.

Once 5 trials have been completed, continue and repeat steps 1-6 for the other temperatures (15oC, 25oC, 35oC & 45oC).

1. Take a paper towel and cut out two circles that are the same size as the base of the petri dishes.

The optimal temperature of Bacillus lichenformis bacterial amylase and Aspergillus oryzae fungal is determined by mixing a starch solution into the bacterial and fungal amylases that are put in four different temperatures (0, 20, 55, 85 degrees Celsius). Then after every two-minutes, ending at the ten-minute mark, a small sample of the starch-amylase mixture is put into a well with a couple drops of iodine to help show the change in starch. This was done because when iodine is exposed to starch it changes color. Based on the color chart given in our lab manuals, the reaction of the amylase to the starch solution will give the starch-amylase mixture in the iodine a yellow color to signify if the presence of solely iodine and/or little starch depending on temperature. This means that the amylase broke down the starch solution because its temperature was optimal. Majority of the results came out black or dark brown therefore the amylase wasn’t put in the proper temperature to break down the starch solution at a faster pace. The temperature that seemed most optimal was at 55 degrees Celsius for both fungal and bacterial because it showed a more brown to yellowish color when put into the iodine. That showed that the amylase was able to break down the starch at a faster rate because it was working at its optimal temperature.

The purpose of this experiment was to determine (1) the reaction rate of an amylase enzyme in starch and (2) the environmental factors that can affect the enzymatic activity. The hypothesis, in relation to the enzymatic activity by variables such as the substrate concentrations, temperature, PH and chemical interactions on the rate of reaction, stated

An experiment was performed to test how temperature variations affect enzymatic activity of the enzyme amylase. The results of the experiment will also determine the optimal temperature of the amylase enzyme. The results of the experiment provide evidence for determining the environments that the enzyme amylase would most likely be present. By determining the possible environments, one can predict what and how environmental factors will affect the enzyme amylase. Two forms of amylase (Bacterial - Bacillus licheniformis and Fungal - Aspergyllus oryzae) were combined with starch molecules at four different temperatures (0⁰, 25⁰, 65⁰, 85⁰ Celsius). The combination of starch and the amylase enzyme resulted in a visual chemical reaction that was recorded. The enzyme activity was recorded every two minutes, starting at 0 and ending at 10. The start time 0 served as the control group of the experiment. The results concluded that both bacterial and fungal amylase has an optimal temperature around 65⁰C. This was possible to determine by recording the color change of the spot plate wells. Amylase catalyzes efficiently at its optimal temperature which resulted in yellow spot plate wells. Enzymatic activity decreased when the temperature was less than 65⁰C, resulting in a green-brown well. The green wells indicated that starch wasn’t broken down completely and was still present. Temperatures greater than 65⁰C resulted dark-green wells which resembled the denaturing of

An enzyme also known as a protein, is a biological catalyst which speeds up chemical reactions by lowering the activation energy to increase the rate in which the reaction occurs. The enzyme used was amylase, which breaks down starch molecules into maltose. PH, substrate concentration, salt concentration, and temperature. When enzymes reach a low temperature, the activity is slowed down of molecule movement, but the enzyme is not destroyed. Once enzymes are placed in optimal temperatures once again, it will restore its activity to a normal rate. When enzymes reach too high above optimal temperature, the enzyme is denatured and cannot be restored. In the experiment performed the activity of breaking down starch in fungal and bacterial amylase was being tested at a range of temperatures and time. The fungal and bacterial amylase work best at optimal temperature. Amylase will function best at sixty degrees Celsius at 10 minutes when starch had been one hundred percent hydrolyzed. Hydrolyzed is the breakdown of molecules through addition of water. The experiments independent variables were the time, temperature and enzyme used. The dependent variable was the enzyme activity that broke down the starch into maltose. The controlled variables were the temperature baths, the iodine drop amount, the mixture drop amount, and location of experiment. The control group was the zero minutes without amylase at

4. Place 25 mL of the low concentration tobacco solution into the “low concentration” petri dish. 5. Place 25 mL of the medium concentration tobacco solution into the “medium concentration” petri dish. 6.

4.Measure 35mL of warm water and add them into each of the 4 test tubes at about roughly the same time. It is essential that the water is warm. Do not seal the test tube.

During these experimental procedures, the implication of multiple different temperatures on fungal and bacterial amylase was studied. In order to conduct this experiment, there were four different temperatures used. The four temperatures used were the following: 0 degrees Celsius, 25 degrees Celsius, 55 degrees Celsius, and 80 degrees Celsius - Each temperature for one fungal and one bacterial amylase. Drops of iodine were then placed in order to measure the effectiveness of the enzyme. This method is produced as the starch test. The enzyme was tested over the course of ten minutes to determine if starch hydrolysis stemmed. An effective enzyme would indicate a color variation between blue/black to a more yellowish color towards the end of the time intervals, whereas a not so effective enzyme would produce little to no change in color variation. According to the experiment, both the fungal amylase and bacterial amylase exhibited a optimal temperature. This was discovered by observing during which temperature and time period produced a yellow-like color the quickest. Amylase shared a similar optimal temperature of 55 degrees Celsius. Most of the amylases underwent changes at different points, but some enzymes displayed no effectiveness at all. Both amylases displayed this inactivity at 0 degrees Celsius. At 80 Celsius both the enzymes became denatured due to the high temperatures. In culmination, both fungal and bacterial amylase presented a array of change during it’s

Placed it to the shaking incubator at 37˚C to prewarm. Heat shocked the cells for 60 seconds in 42°C water bath. Added 950 µl of warm (37°C) SOC medium and incubated with shaking (160-225rpm) for 50 minutes at 37˚C. Used two LB plates. Plate 100 µl of each transformation on a separate LB agar plate containing carbenicillin which is the antibiotic, IPTG to induce expression of lacZ and X-gal a substrate for β-galactosidase. The plastic L-spreader was used to distribute the bacteria.

Effect of varying Temperatures on Enzymatic Activity of Bacterial and Fungal Amylase and hydrolysis of Starch

The Effect of different Temperatures on the reaction rate of Amylase Introduction: Amylases are enzymes that breaks down starch which accelerates the hydrolysis of glycosidic bonds in polysaccharides (S. Sivaramakrishnan et al., 2006). They bond with certain substrates to effectively increase the reaction rate of a chemical experiment. Most enzymes are very specific, as in they would only bond with a distinct substrate for it to cause a certain reaction. This can be described with the key and lock analogy. Only one type of key, certain amylase, will be able to open a particular lock, substrate.

In this lab, I tested in which conditions starch synthesis will take place and how the temperature of the reaction mixture influences starch synthesis. My main goal for this experiment is to learn about the basic principles of enzyme functions, and how their environment around them can influence them.

Enzymes are catalysts that regulate the chemical reactions that occur in a cell. By lowering the activation energy, they can increase the rate of reactions. Enzyme amylases such as Bacillus Licheniformis and Aspergillus Oryzae split starch polymers into smaller subunits called monomers. The lab displayed how divergent temperatures affect the activity of the enzyme amylase and reveal the optimal temperature for the fungal and bacteria amylase. To set up the experiment, eight test tubes are necessary. Four of the test tubes will be labeled for the bacteria and the remaining four will be labeled fungi. Each test tube will be labeled for a specific temperature (0⁰, 25⁰, 55⁰, 85⁰ Celsius) and another 4 for the starch solution. Two spot plates are required which will be used to record and analyze each reaction every two minutes by adding iodine to the well of the spot plate. After gathering the information you will need to determine what color represents the greatest reaction can be determined by the color (Black – Yellow). It is important to collect and compare every group’s data in order to obtain accurate results. After going over all the results, one can conclude that the best temperature for the reaction to occur in this solution is at 55⁰ Celsius which would be the enzyme’s optimal level. Knowing what temperature works best for enzymes allows for a faster and more efficient use of these reactions. Without enzymes, the reactions would be too slow to keep the cells