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An investigation into the effect of pH on the starch hydrolysis of fungal and bacterial amylase

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Introduction

An investigation into the effect of pH on the starch hydrolysis of fungal and bacterial amylase. Hypothesis The optimum pH of fungal amylase is lower than the optimum pH of bacterial amylase. Variables In order to keep results reliable it will be important to ensure that there is only variable. This will be pH. This means that all other things must be kept constant. In order to achieve this certain precautions need to be taken. Temperature will be maintained by putting the agar plates in an incubator set at a constant of 20�C; this will be checked with the use of a thermometer. This is important because an increase in temperature will increase kinetic energy therefore speeding up the rate of reaction. High temperatures could even denature the amylase. Low temperatures will decrease the rate of amylase activity because the substrate and enzyme molecules have less kinetic energy and move more slowly, collide less often and react more slowly. The same concentration of bacterial and fungal amylase will be used (0.1%). It is known that a higher concentration will cause a faster reaction rate, this is because there will be more active sites for the substrate to react with. To keep the concentration at a constant the enzyme will be weighed out on a top pan balance to an accurate degree, and dissolved in 100cm� of distilled water. The rate at which the enzyme functions depends partly on the concentration of enzyme molecules. The greater the concentration of amylase, the more active sites there are and so the more chances there are of collisions between the substrate and active site. The same volume of buffer and amylase needs to be used in each hole on the agar plate. A 1cm� syringe will be used, as this can dispense within a 0.005cm� accuracy. Also in order to keep the diameter of the hole constant a cork borer of diameter 5mm will be used. ...read more.

Middle

None of colourless areas of hydrolysed starch overlapped each other after this length of time, so I have decided to leave the plates for 24hours, to maximize the volumes of starch hydrolysed, in order for there to be more reliable and notable differences between the results at different pH values. -I felt the use of a compass as a measuring tool was slightly unreliable, and did not feel that it was giving the most accurate measurements. Therefore I am now planning to use dividers as I think these will be easier to use and will give more accurate measurements. Revised apparatus * 10 starch agar plates (1 g 100cm3) * dividers * 0.1 g 100cm3 bacterial amylase * 0.1 g 100cm3 fungal amylase * stopwatch * pH 2, 4, 6, 8 and 10 buffers * cork borer * iodine * 3 syringes (1cm�) * ruler Introduction Amylase is an enzyme and is therefore classed as a biological catalyst. A catalyst is a chemical agent that speeds up the rate of metabolic reactions, by lowering their activation energy (which is the energy required to start a reaction), without being used up in the process. It does this by changing the pathway which the reaction takes. Enzymes are site specific, meaning they can only act on a particular substrate. All enzymes are proteins. They have a three dimensional (tertiary) structure that are specific to their function. The structure is held together by hydrogen bonds, ionic bonds and disulphide bridges. During the reaction some bonds are broken and some are made, this allows the reactants to be changed into products. On the surface of the enzyme is a depression called an active site. This is formed from the residues of a small number of amino acids and has a very specific shape that is complimentary to the shape of the substrate. Any factor that changes the tertiary structure of the whole protein can alter the shape of the active site and alter the activity of the enzyme. ...read more.

Conclusion

Some of the sets of repeats show little variability and therefore can be considered reliable; for example the diameter of starch hydrolysed by fungal amylase at pH 6 has a mean diameter of 21.08mm and all repeats are within 2.8%. All other pH values tested for fungal amylase have repeats within 4%. With bacterial amylase at pH 2 the mean diameter of starch hydrolysed was 16.42mm and all repeats are within 3.4%. However in other bacterial amylase agar plates of different pH, there is a slightly greater variability, indicating that the results may not be completely reliable. For example for with bacterial amylase at pH 10 there was mean diameter of 18.00, with repeats within 5.26%. None of the repeats, for any of the pH values were beyond 5.26% of the mean, but to increase reliability, more repetitions would need to be made for each pH value for both fungal and bacterial amylase. The variability of the results can also seen on graph 1 where the error bars provide the largest and smallest volumes of starch hydrolysed within each agar plate. The greater the size of the error bar, the less reliable the results are. In the graph none of the bars overlap one another this means that there definitely is some variability in my results, however none of the bars, or the data show any significant outliers. In table 3 and table 4 possible anomalies are highlighted in red, these are the results furthest away from the mean. However it is not believed that these results would have any significant effect on the overall results, and they have been included in all calculations. The reason that these anomalies may have occurred is due to lack of control of variables, as this causes the variation in raw results which leads to anomalies. Despite trying to control all possible variables there are a few areas within the plan which could have lead to inaccuracy in the results. Firstly when using the 1cm� syringes, to measure 0.05cm� of buffer and 0.05 cm� of amylase, it was important to make sure that ...read more.

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