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The effect of pH on an enzyme - Fungal amylase.

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Introduction

THE EFFECT OF pH ON AN ENZYME - FUNGAL AMYLASE. Background information: Enzymes have five important properties: * All enzymes are proteins. * They are specific in their action. * They can be used over again. * They are destroyed if the temperature is too high. * They are destroyed if the pH is too high or too low depending on the enzyme used. Enzymes are substances that act like catalysts i.e they increase the rate of the chemical reaction without themselves being used up in the reaction. Enzymes are all globular proteins i.e their molecules are round in shape. The substance to which the enzyme combines to is known as the substrate. The surface on the enzyme molecule to which the substrate combines is known as the active site. The shape of the active site is complimentary to the shape of the substrate. The enzyme speeds up the process of conversion of substrates into products. Although the enzyme combines with the substrate for a short time, the enzyme and the substrate split apart afterwards releasing the enzyme. Thus the enzyme is not used up in the process and so it can still be used over again. Enzyme + Substrate-Enzyme substrate -Enzyme product - Enzyme+Product complex complex E + S - ES - EP - E + P Starch: Starch is the storage carbohydrate in plants and in animals starch is used as an energy store. Animals obtain their starch from a variety of plant sources. Starch is a polymer of alpha glucose monomers and is a mixture of amylose and amylopectin.The alpha glucose monomers are joined together by 1,4 and 1,6 glycosidic bonds. Amylose is a single unbranched polymer chain of 500 to 2000 glucose subunits with only 1,4 glycosidic bonds. Amylopectin is branched with both 1,4 and 1,6 glycosidic bonds. When fungal amylase acts on starch it results in the formation of mono-, di- or tri- saccharides. ...read more.

Middle

Amount of starch(mls) Amount of amylase(mls) Test for starch Time(seconds) 1 5 5 5 Positive 2400 3 5 5 5 Negative 60 4 5 5 5 Negative 60 5 5 5 5 Negative 60 6 5 5 5 Negative 30 7 5 5 5 Negative 60 9 5 5 5 Positive 2400 2.5mls of Fungal amylase TEST 1 pH Amount of pH(mls) Amount of starch(mls) Amount of amylase(mls) Test for starch Time(seconds) 1 5 5 2.5 Positive 2400 3 5 5 2.5 Negative 150 4 5 5 2.5 Negative 90 5 5 5 2.5 Negative 60 6 5 5 2.5 Negative 30 7 5 5 2.5 Negative 300 9 5 5 2.5 Positive 2400 1mls of Fungal amylase TEST 1 pH Amount of pH(mls) Amount of starch(mls) Amount of amylase(mls) Test for starch Time(seconds) 1 5 5 1 Positive 2400 3 5 5 1 Negative 240 4 5 5 1 Negative 90 5 5 5 1 Negative 90 6 5 5 1 Negative 60 7 5 5 1 Negative 300 9 5 5 1 Positive 2400 Procedure: When I started the experiment I made sure that I had labelled all my pipettes carefully so that I could use them again without mixing them up. Then I placed my test tubes in the test tube rack so that I wouldn't accidently drop any of them. I then measured out 10mls of the buffer solution using the pipette and the pumpette. I also made sure that I measured out the 10mls as accurately as I possibly could without letting any air bubbles into the pipette. Then I transferred the 10mls into the test tube and added 0.5mls of the fungal amylase that were freshly made just before I started the experiment. I also took the same precaution as I did when I measured out the buffer solution but I also made sure that I did not spill any of the amylase on my hands or on the table top. ...read more.

Conclusion

But if I had been given more time then I would have tried adding the starch and the pH before adding the amylase. Then I could compare the results get for this with the results that I already have to see if this would cause any difference. The most important limitation to this was in making an accurate judgement of the final end point of the reaction. After the end of some reaction the iodine appeared to have a dark brown colour and at the end of some it had a light brown colour so the exact end point was hard to judge. When I did the experiment I only started timing after I had completely added my 10mls of starch and this is not very accurate because the enzyme would have started reacting with the starch as soon as the first drop was added. So I can't completely say that the time I got for the experiment is very accurate because some of the starch would have already been converted to maltose even before I started the stop clock. Since there was only a limited amount of time given to me to do the experiment , I only did pH 1,3,4,5,6,7 and 9 but if I had been given more time then I could have tested a wide range of pH eg: pH 4.5, 6.5,8 etc to see exactly at which point the amylase works best. I could have also change the concentration of the starch if I had more time. But overall with the work I have done and by looking at my results I can draw a firm conclusion that my hypothesis is right and that the enzyme does get denatured if the pH is too acidic or too alkaline and the time taken for the reaction to be completed increases. The experiment also shows that there is a certain pH at which enzymes will work but if the pH is too extreme the enzyme gets denatured. But clearly repeating my experiment would further give me more reliable results. 1 ...read more.

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