Hydrolysis of starch for fungal amylase Aspergillus Oryzae and bacterial amylase Bacillus Licheniformis at different temperatures.
Maydelis Perez PI 4593042
Florida International University
BSC 1010L, section UO9, March 6, 2013
Abstract Enzymes are very specific protein because they contain one active site on their surface that enable the substrate to bind to the enzyme and form the enzyme substrate complex and then release the products. The principal function of enzymes is to increase the rate of the chemical reactions. Enzymes have a set of conditions at which they work perfectly; this is known as optimal condition. Fungal and bacterial amylase are the
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Fungal Aspergillus oryzae and bacterial Bacillus licheniformis amylase are the two types of enzymes that will be used during this lab to test the ability of amylase to break down starch ,a polysaccharide present in our daily diet, into simpler sugars , maltose (Smith,et al,2012).
Bacterial amylase is found in different habits for example in human saliva, for this reason is easy to infer that bacterial amylase would break down the starch better and faster than the fungal amylase. According to this information amylase activity going to change higher or lower according to the temperature but it will be faster in bacterial than in fungal, 100% hydrolysis going to occur between 40°c to 60°c because this is the habitat temperature for these species and this means that is the optimal temperature, but in bacterial occur in less time than in fungal because are found in human saliva were chemical digestion begins.
Methods The principal objective of this study was to determine how temperature affects the activity of amylase enzyme and the optimal temperature for fungal and bacterial amylase. In order to make this experiment possible we needed fungal Aspergillus oryzae amylase and bacterial Bacillus licheniformis amylase; 5 ml of 1.5% starch were placed in one test tube
For enzymes to be useful in a reaction, the substrate needs to bind with the enzymes active site. The active site is specific for a
There are many types of enzymes and each has a specific job. Enzymes are particular types of proteins that help to speed up some reactions, such as reactants going to products. One of them is the amylase enzyme. Amylases are found in saliva, and pancreatic secretions of the small intestine. The function of amylase is to break down big molecules of starch into small molecules like glucose; this process is called hydrolysis. Enzymes are very specific; for example, amylase is the only enzyme that will break down starch. It is similar to the theory of the lock
Enzymes are types of proteins that work as a substance to help speed up a chemical reaction (Madar & Windelspecht, 104). There are three factors that help enzyme activity increase in speed. The three factors that speed up the activity of enzymes are concentration, an increase in temperature, and a preferred pH environment. Whether or not the reaction continues to move forward is not up to the enzyme, instead the reaction is dependent on a reaction’s free energy. These enzymatic reactions have reactants referred to as substrates. Enzymes do much more than create substrates; enzymes actually work with the substrate in a reaction (Madar &Windelspecht, 106). For reactions in a cell it is
Note that the enzyme remains unchanged so that more of the some substrates can react.
• Enzymes bind to a substrate (anything that needs to be changed into something else [molecule, protein etc.])
Enzymes are biological catalysts, which accelerate the speed of chemical reactions in the body without being used up or changed in the process. Animals and plants contain enzymes which help break down fats, carbohydrates and proteins into smaller molecules the cells can use to get energy and carry out the processes that allow the plant or animal to survive. Without enzymes, most physiological processes would not take place. Hundreds of different types of enzymes are present in plant and animal cells and each is very specific in its function.
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
“Enzymes are proteins that have catalytic functions” [1], “that speed up or slow down reactions”[2], “indispensable to maintenance and activity of life”[1]. They are each very specific, and will only work when a particular substrate fits in their active site. An active site is “a region on the surface of an enzyme where the substrate binds, and where the reaction occurs”[2].
Bacterial amylases operate at higher temperatures than do fungal amylases. Fungal amylases react rapidly at lower temperatures; fungal amylases are used as an agent for alcohol fermentation for grain (Underkofler et al, 1958). Fungal amylases is said to be denatured – change shape (Alberte et al, 2012), at high temperatures above 60° C and bacterial amylases on the other hand are stable and show little denaturing at temperatures up to 85°C 3 The question answered by the experiment is if the temperature is not within the range of the enzymes (fungal and bacterial amylase) optimal temperature (higher temperature) then will the enzymes denature and if the enzymes are placed in lower temperature from optimal the activity then will it slow down enough to stop all reaction, meaning each enzyme will not be operating efficiently. Knowing about a bacterial amylases and fungal amylases optimal temperatures are important for knowing which food products and industrial products it can be used on to conserve the product because then the producer knows about which products it can be incorporated into depending on the temperature it is manufactured at.
Organisms cannot depend solely on spontaneous reactions for the production of materials because they occur slowly and are not responsive to the organism's needs (Martineau, Dean, et al, Laboratory Manual, 43). In order to speed up the reaction process, cells use enzymes as biological catalysts. Enzymes are able to speed up the reaction through lowering activation energy. Additionally, enzymes facilitate reactions without being consumed (manual,43). Each enzyme acts on a specific molecule or set of molecules referred to as the enzyme's substrate and the results of this reaction are called products (manual 43). As a result, enzymes promote a reaction so that substrates are converted into products on a faster pace (manual 43). Most enzymes are proteins whose structure is determined by its sequence of its amino acids. Enzymes are designed to function the best under physiological conditions of PH and temperature. Any change of these variables that change the conformation of the enzyme will destroy or enhance enzyme activity(manual, 43).
Enzymes are central to every biochemical process. Due to their high specificity they are capable of catalyzing hundreds of reactions that signifies their vast practical importance.
The effects of temperature on fungal amylase Aspergillus oryzae, and bacterial amylase, Bacillus licheniformis ability to break down starch into maltose was studied. The study determined the optimal temperature the Aspergillus oryzae and Bacillus licheniformis was able to break down the fastest. The starch catalysis was monitored by an Iodine test, a substance that turns blue-black in the presence of starch. Amylase catabolizes starch polymers into smaller subunits. Most organisms use the saccharide as a food source and to store energy (Lab Manual, 51). The test tubes were labeled with a different temperature (0°C, 25°C, 55°C, 85°C). Each test tube was placed in its respective water baths for five minutes. After the equilibration process, starch was placed in the first row of the first row of the spot plate. Iodine was then added to the row revealing a blue black color. The starch was then added to the amylase. After every two minute section a pipette was used to transfer the starch-amylase solution to place three drops of the solution into the spot plate row under the corresponding temperature. Iodine drops was placed in the row. Color changes were noted and recorded. The results showed Aspergillus oryzae was found to have an optimal temperature between 25°C and 55°C and Bacillus licheniformis was found to have an
Enzymes are very specific in nature, which helps them in reactions. When an enzyme recognizes its specific substrate, the
Different Salt Stresses on pH Stressed Culture With Maximum α-amylase Activity: The salt at different amount was given to Bacillus amyloliquefaciens to act as supplement for amylase production. In present study different amount like 1%, 1.5%, 2% and 2.5% were used and produced amylase was studied followed by DNS enzyme assay method.