The Effect of pH on Fungal and Bacterial Amylase

The Effect of pH on Fungal and Bacterial Amylase
Amylase is a digestive enzyme systematized as a saccharidase (an enzyme that splits polysaccharides). It is mainly a composition of pancreatic juices and saliva, needed in order to break down long chain carbohydrate insoluble molecules (such as starch) into smaller soluble units. Enzymes are biological catalysts that speed up specific chemical reactions and are made up of globular proteins made and used by our bodies in human use. Enzymes are not only used in human natural processes they are also used in manufactured and industrialised areas such as in the making of bread, cheese, yoghurt, wine making etc. They are used in various forms of chemical reactions at temperatures suitable for in living organisms of between 4 and 40 C . Enzymes are vital for life as their deficiency in a reaction would cause very slow reactions to maintain life. The chemical(s) which an enzyme works on is called the substrate. An enzyme combines with the substrate to form a temporary enzyme-substrate complex.
The higher the amount of enzyme to substrate increases the chances of reactions. When the reaction occurs the substrate is broken down into products and yet the enzyme does not get used up and is free to work its cycle again. There are certain factors which affect the rate at which these enzymes can work and these are temperature, substrate concentration, enzyme concentration and pH. PH is a scale of 1-14 in which it contains chemicals which are acids and alkalis. It is scale which is determines the strength of the acid or alkali (pH 1 being strongest acid, pH 14 being strongest alkali and pH 7 being neutral). Acids are chemicals between pH 1-6 which donate H+ ions in aqueous solution and alkalis take away OH- ions away in aqueous solution. Initially enzyme amylases breakdown starches into substances that can be absorbed by the body for use. Ptyalin is the name given to the amylase present in saliva where the digestion of starch happens first in the mouth. The function of amylase enzymes are to catalyse and hydrolyse of 1,4 and 1,6 glycosidic linkages of polysaccharides (amylose and amyl pectin) into a variety of products including maltose, dextrin and D-glucose etc. Amylases are obtained from animal, bacterial, fungal and plant sources such as barley, malt and fungi called Aspergillus oryzae. There are a few forms of amylases. Eukaryotes are membrane bound nuclei cells with some membrane bound organelles. They carry out the processes of life. They are classified as animals, plants, protoctists and fungi. Fungi are a huge group of organisms that are ranged in size from unicellular yeasts to large toadstools and puffballs etc and are found in a range of habitats; aquatic and terrestrial. They are moulds that grow on dead damp organic matter such as bread, non living vegetation etc. Prokaryotes are non membrane bound cells and do not contain nuclei and have a few of the organelles found in eukaryotes. They are commonly made up of organisms such as bacteria. These bacteria species are the smallest and oldest form of organisms with a cellular structure. They come in a range of sizes but are much smaller than eukaryotes. They live in environments of soil, air, dust, water and on animals and plants. Hypothesis My hypothesis is that: ?The greater the pH the higher the activity of bacterial amylase in starch hydrolysis than fungal amylase?. Fungi and bacteria are both forms of amylases and are used in feeding processes. As mentioned above bacteria are prokaryotes they are very small organisms with no true nucleus and a unicellular form and occur by clumping together in characteristic patterns forming chains but the cells can also be individual cells and have to be seen under microscopes as they are very small. They can be found in most environments such as dust, air, soil, water and living materials. Together with fungi they play an important role in vital activities with organisms in decaying of recycling of nutrients from organic matter. Some bacteria can inhibit in many environmental extremities of pH 1-11 in order to maintain constant cytoplasmic pH ranges. Fungi are eukaryotes which are much bigger cells with membrane bound nuclei and organelles. They compose of mildews, rusts, smuts, puffballs, mushrooms, yeasts, stinkhorns, toadstools etc. They grow on dead matter, parasites or dissolved nutrients through their cell walls. It has a fungal body made up mycelium with parts of hyphae( fine, thin, long threads) which form even longer branching chains of fungi cells. Fungi usually grow best at slightly acidic pH of around 5-7 and so can reproduce at this range. I have chosen bacterial amylase that work best at higher pH values because they can be found in a range of different habitats and if this is so it can tolerate the extremities in pH and so would have a larger optimum pH range. Generally any enzyme works best and faster at a certain optimum pH range, under constant temperature. Some work in more acidic conditions such as pepsin in the stomach will need to work best at pH 2 as it is working with hydrochloric acid, and others in alkaline conditions. Increase in temperature creates a bigger kinetic movement across the molecules and so having a greater chance of them colliding with each other. However enzymes also work at optimum temperatures before they become inactivated (denatured). If one is working with pH the temperature has to be kept constant as it is a factor that can also affect enzymes. The optimum pH is the maximum rate of reaction that occurs in the range. When pH is altered below or above the optimum the activity is decreased or becomes denatured. As pH is reduced it becomes acidic and an increase in H+ ions which increase the amount of positive charges. Alterations in pH change the ionic charges of the acidic and basic groups and therefore mutate the R-groups in the amino acids which disrupt the ionic bonding which help stabilise the specific shape of the enzyme. The pH change therefore leads to the alteration of the enzyme shape and also the active sites in which substrates are broken down, causing the enzyme to denature. [image] A graph showing enzyme activity against pH I am going to investigate the effect of pH of the enzyme activity of fungal and bacterial amylases using various pH and using it to hydrolyse starch and testing it using potassium iodine solution for starch present under timed conditions with these two different amylases separately. The presence of starch can be discovered using iodine dissolved in potassium iodide which is a orangey brown colour and changes to a dark blue-black colour when starch is present. The experiment is to investigate the effect of pH on the two types of amylases on its activity. The relative activity of the amylases is noted by the time taken for the starch substrate to be broken down resulting in a colour that is no longer a blue- black colour when tested with the iodine solution. This is known as the achromatic point. Key variables In order for the experiments to be fair and reliable each time the following must be taken in consideration: starch concentration and volume will be the same in all experiments. Amylase concentration and volume will be the same in all experiments. The same concentration and drop size of iodine will be used on the spotting time to determine starch presence. Buffer solutions would be used to avoid any changes in pH differences The iodine solution would be used as a guide to determine the end point colour. Other volumes of liquid would measured accurately using measuring cylinder and pipettes. All this would be timed using a stop watch Apparatus 0.1% fungal amylase and bacterial amylase Potassium iodine solution 1.0% starch solution White spotting tile 10 clean test tubes Test tube rack Buffer solutions ranging from pH 4-8 Pipette Measuring cylinder And stopwatch. Method Take a test tube rack and place five clean test tubes each for the fungal amylase and bacterial amylase and place a sticker with a different pH of 4-8 so you do not get confused. Prepare the buffer solutions by diluting the acid or alkali of certain concentration (mol dm ) with 100ml of water. Use a universal indicator to check the pH. Pipette 1cm of either fungal or bacterial amylase decide which one you are going to do first in each of the five test tubes. Then take a pH solution of 1 cm and place in one of the test tubes with its correct label. Time this for ten minutes. Whilst waiting place equal drops of potassium iodine solution in different places on the spotting tile. After ten minutes, add 5 cm 1.0% starch solution. Now for every thirty seconds for five minutes, pipette a bit of the solution and place it on a clean spot of iodine solution and record the results. + is dark blue-black, +/- if dark brown, and ? is no colour change. Repeat this with the other pH solutions and the other amylase enzyme. Risk assessment/ Safety precautions Amylase: It is brought in powdered form and so can causes respiratory problems if inhaled in a poorly ventilated room. It may cause risk of eye damage if contact with eye. On skin contact can causes cracked skin or eczema. To avoid this wearing eye protection and gloves to be safe. Buffer solutions: Handle with care wash with water if comes in contact with skin. Protect from eyes as it can be irritant. Avoid by wearing gloves and eye protection. Iodine in potassium iodine solution: It can be harmful if inhaled, swallowed or absorbed by skin. If absorbed by skin or comes in contact with eyes it can be irritant. It causes gastrointestinal discomfort if swallowed or respiratory problems if inhaled. Washing spotting tiles thoroughly under running water and clean any spillage.

The Effect of pH on Fungal and Bacterial Amylase 7.6 of 10 on the basis of 3983 Review.