Name: Swara K Patel
Panther ID: 6134894
Lab partners: Ana Araujo, Monique Salcie, Gabriel
The Effect of Temperature on Enzymatic Activity of Bacterial and Fungal Amylase
What are enzymes? Enzymes are the biological catalysts that changes the rate of the reaction without being consumed by the reaction. They increase the rate of reactions by lowering its activation energy i.e. the minimum amount of energy required for the chemical reactions to occur. They are proteins composed of long chains of amino acids and form complex structures. Most enzymes are three-dimensional in structure and acts on reactant which is known as substrate. The substrates are recognized by their amino acid sequences. The enzyme binds to the active site of the substrate which is usually recognized by few of the amino acids and thus they form the enzyme-substrate (ES) complex which is held by weak bonds. The enzyme which have the similar shape to that of substrate can bind to its active site. This specificity is known as “lock and key model.”. Here the substrate is the key and the enzyme is the lock in which it fits. After the substrate binds to the enzyme it reshapes itself so that the enzyme could fit in snugly into the substrate. This potential of enzymes is known as “induced fit model.”
There are many enzymes present and each of the enzyme has a specific set of condition which is known as its optimal conditions. These conditions are mainly based on the environment that the enzymes are found in. And these conditions; such as pH, temperature, salt concentration, substrate concentration, as well as presence of inhibitors, activators and cofactors affect the enzymatic activity. For example; the enzyme denatures at a temperature higher than its optimal conditions i.e. changes its shapes and hence preventing the substrate binding. The way enzymes respond to temperature is fundamental to many areas of biology. Until recently, the effect of temperature on enzyme activity has been understood in terms of raised temperature increasing activity and, at the same time, causing activity to be lost by denaturation (Daniel et.al, 2010) This observed “optimal temperature” is not an intrinsic enzyme property, since it arises from a mixture of thermal properties, and from assay duration (Daniel et.al,2008)
The enzyme used in this experiment is amylase. It is an enzyme which catabolizes starch polymers. It breaks starch molecules into short oligosaccharides, maltriose and maltose. Amylase is important and indispensable enzyme that plays a pivotal role in the field of biotechnology. Amylases are broadly classified into ?, ?, and ? subtypes, of which the first two have been the most widely studied (Gopinath et.al, 2017). Both starch and amylase are commercially being used in production of syrups and other food products and most organisms use these saccharides as a food source and to store energy.
There are two different amylases used in this experiment namely bacterial amylase and fungal amylase. The bacterial amylase was extracted from Bacillus licheniforms and the fungal amylase was extracted from Aspergillus oryzae. B. licheniforms was particularly used for this experiment because the advantage of using -amylses is that it has decreased risk of contamination, increased diffusion rate and decreased cost of external cooling. (Lin et.al,2010). Aspergillus oryzae was selected because Aspergillus species isolated from various seeds were screened for their ability to produce amylase. A selected strain showed the highest amylase activity (Anupama et.al, 2007).
The independent variable in this experiment is the temperature of amylase and dependent variable is the digestion of starch. To measure this colour change iodine is used as an indicator. When iodine reacts with starch it gives blue-black colour which shows that there is no breakdown of starch (substrate) and as the colour gets lighter it indicates the digestion of starch by amylase.
If the colour of the starch changes from dark-blue to yellow, then is shows that there was breakdown of starch at that particular temperature because every enzyme has an optimal temperature at which it shows maximum activity. And this is the purpose of this experiment to find out at which temperature the amylase is most effective at digesting the starch?
Firstly, we need an experimental setup. Then, we placed spot plates on the paper napkins and wrote temperature (in degree Celsius) on the top (0, 25, 35, 55, 85) and the time (in minutes) on the side (0, 2, 4, 6, 8, 10). Then take 4 test-tubes and label them according to the temperature and the enzyme source (B- bacterial and F – fungal) and the group number. Again, we took 4 test-tubes and labelled those according to the temperature and enzyme source (B or F), our group number and the letter S (for starch solution) and then added 5mL of 1.5% starch solution into the test-tubes labelled S.
After this we prepared the amylase test-tubes and added 1 mL of amylase into the tubes that did not contain starch. We took the tubes assigned for the bacterial amylase and placed them all into their respective temperature water bath for 5 minutes. We added 2 drops of iodine in the 0-minute row. Without removing the tubed from the bath, we transferred a few drops of starch solution in the dish for the 0-minute row. Then after, we poured the remaining solution of starch into the amylase tubes. Then observed the results in the given time-period.
important as they
354965245745743585116205Temp (C) 0 25 55 85
Time(minutes) 0 4.001.26 3.67 1.51 3.83 0.98 4.17 1.60
2 2.671.37 2.001.26 2.000.89 3.000.89
4 2.331.03 2.001.10 1.830.75 2.831.17
6 2.171.17 2.001.10 1.830.75 3.671.03
8 2.331.03 2.001.10 2.000.63 3.831.17
10 2.171.17 1.831.17 1.830.75 3.671.51
Optimal temperature 55C
Time to 100%Hydrolysis at Optimal Temp. 10 minutes
The optimal temperature of bacterial amylase is 55C. At 0C there was no colour change as all the columns were black-blue colour observed and this indicates that there was no breakdown of starch. At 25C a slight variation was observed as the blue-black colour changed to light brown but not much was observed and that is the reason that 25C was not the optimal temperature. At 85C again no colour change was observed. The highest variation in colour was observed at 55C and hence it is the optimal temperature for bacterial amylase to break down starch molecules.
324485184150667385115570Temp (C) 0 25 55 85
Time(minutes) 0 4.171.33 4.001.55 4.670.82 5.000.00
2 3.001.41 2.671.21 2.831.33 4.830.41
4 3.501.05 2.831.17 2.501.52 4.830.41
6 3.331.03 3.171.47 2.501.05 4.830.41
8 3.001.41 3.001.55 2.501.05 4.830.41
10 3.171.17 3.171.47 2.671.21 4.830.41
Optimal Temperature 25
Time to 100%Hydrolysis at Optimal Temp 10 minutes
The optimal temperature for fungal amylase is 25C. At 0C there was no colour change observed as all the columns were blue-black colour and it shows that there was no breakdown of starch at that temperature. At 55 the colour changed from blue-black to dark brown which indicates that there is a slight breakdown of starch. At 85C there was no colour change observed. The highest colour change was observed at 25C and hence maximum breakdown of starch occurred at this temperature.
It appears that for bacterial amylase the optimal temperature was 55C as the enzyme showed maximum activity at that particular temperature. At 0C there was no change