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Comparing the denaturation rate of fungal and bacterial amylase.

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Introduction

Comparing the denaturation rate of fungal and bacterial amylase. Plan Amylases are widespread enzymes which hydrolyse starch to maltose. They are often found in two forms, ? amylase which degrades starch molecules into fragments 10 glucose residues long and ? amylase which breaks down these into maltose made up of two glucose molecules. Both work by hydrolysis adding one molecule of water across the glycosidic link. Hypothesis My hypothesis is that bacterial amylase will work more efficiently at higher temperatures than fungal amylase. Both bacteria and fungi use amylases in their basic method of feeding. Bacteria are prokaryotes which means they are very small and have no true nucleus. They are unicellular but occur together in vast numbers as large groups or entirely separate cells. Being found almost everywhere in air, soil, water and in living things they are of great ecological and economic importance. Many bacteria cause decay and with fungi, facilitate the recycling of nutrients. Bacteria can grow well in a wide variety of conditions and whilst temperatures of 25-450C are most favourable there is a very wide range with some able to continue to grow slowly near to 00C and others able to survive hot springs above 800C. On the other hand fungi are eukaryotes which means they generally have larger cells and have membrane bound organelles. Fungi comprise the moulds, yeasts, mildews, mushrooms, puffballs and rusts. They can be saprophytic, feeding on dead organic matter or parasitic. Fungi consist of a fungal body, the mycelium which is made up of fine threads called hyphae. In a specialised part of the mycelium, spores are produced in vast numbers and dispersed. Moulds which are multicellular fungi, grow best at temperatures of about 300C, their growth is slowed at lower temperatures. In trying to rid foods of moulds the food is heat treated at 60-700C. In comparison bacteria are heat treated to 1000C or more before they are killed off.

Middle

At this point I recorded the time taken. I repeated this experiment for 10mins and 15 mins at each of the temperatures tested (40,50,60,70,80 and 900C). I then began again and repeated exactly the same procedure with the fungal amylase solution. I found that the end point was not always clear so I recorded three different iodine colours to help me analyse the data later. These were, a definite blue/black colour, a dark brownish red and a light reddish brown which matched the original iodine drops. As a control experiment three tests were made in an identical manner with fungal and bacterial amylase except no temperature treatment was given to the enzymes before testing. UA007544 . Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide 31 RESULTS BACTERIAL AMYLASE Treatment Temp 0C Time of treatment (s) 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 40 300 + + +/- +/- +/- - 600 + + + + +/- +/- +/- - 900 + + + + + +/- +/- - 50 300 + + + + + + + +/- +/- +/- - 600 + + + + + + + + + + +/- - 900 + + + + + + + + + + +/- - 60 300 + + + + + +/- +/- - 600 + + + + + + + +/- +/- - 900 + + + + + + + + + +/- +/- - 70 300 + + + + + + +/- +/- - 600 + + + + + + + +/- +/- - 900 + + + + + + + + + +/- +/- - 80 300 + + + + +/- - 600 + + + + + + + +/- +/- - 900 + + + + + + + +/- +/- +/- - 90 300 + + + + + + + + + +

Conclusion

Experimental limitations The most important limitation to this experiment is the difficulty in making an accurate judgement of the final end point. The colour of the iodine changes slowly in some cases. In the slower reactions this was particularly difficult. Whilst some effort was made to distinguish between reactions where the dark brown colour remained after 630s and those where the drops remained blue/black by counting the rate of reaction as zero where there was no sign of change this remained an inaccurate method. The other major limitation involved temperature treatment. Especially at higher temperatures it was difficult to determine the exact time of temperature treatment. The solution itself was checked but inevitably it took longer for the enzyme to reach these higher temperatures so the exact time of treatment did vary. Whilst exactly the same concentration of each enzyme was used the powdered enzymes themselves were rather crude preparations and it is likely that they contain different amounts of active ingredients. This may have accounted for the big difference between the activity of the two enzymes and makes direct comparison more difficult. The overall reliability of the investigation was also limited by time constraints. To cover the full range of temperatures and times of treatment meant that 36 separate experiments were required. This meant unfortunately there was not time to carry out repeat tests. It is evident from the variability of the results especially for bacterial amylase that this was an important limitation. Overall it appeared that this investigation did provide some evidence to support my hypothesis that bacterial amylase would work better over a wider temperature range than fungal amylase. Clearly repeating these tests at least three times would be the most important type of further work which would be needed. If the trends shown in the first set of data are consistent it would also be important to investigate smaller increases in temperature between 50 and 700C. If time was available it would also be profitable to investigate a wider range of fungal and bacterial enzymes possibly from different sources.

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