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Experiment to investigate the effect of Temperature on the enzyme activity of Pectinase

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Experiment to investigate the effect of Temperature on the enzyme activity of Pectinase Aim: To investigate the usage of pectinase in the production of fruit juice at various temperatures by assessing the rate of reaction via measuring the volume of the juice yielded. In order to be able to construct a suitable hypothesis, I must first collect some background information. Introduction: Enzymes Enzymes are complex proteins that are biological catalysts. They are referred Catalyst because a catalyst is any substance which makes a chemical reaction go faster, without being changed or used up in the reaction. Therefore a catalyst can be used over and over again in a chemical reaction. Enzymes are very much the same except that they are made of protein therefore they are easily denatured (loses its protein molecule shape and will not work again) by heat and are also sensitive to pH levels and heavy metal ions. Unlike ordinary catalysts, they are specific to one chemical reaction. An ordinary catalyst may be used for several chemical reactions. Enzymes work best at their optimum temperatures and pH levels. E.g. the enzymes in the human body work at best at 37�C. A molecule of an enzyme, made of globular proteins, is very large but only has a small functional region which is known as the active site. An enzyme will act upon one specific substance or substrate thereby producing a product. Prior to the substrate being changed into a product, the substrate molecule must overcome an "energy barrier." Activation energy must be considered here as it is the amount of energy needed for one substance to collide with another. The smaller the activation energy, the faster the reaction as there will be many substrate molecules with sufficient energy to overcome the activation energy barrier. The rest of the protein hold the molecule together and give the enzyme a specific three dimensional shape, this itself is determined by its tertiary or quaternary structure, held jointly by hydrogen, ionic bonds and di-sulphide bridges. ...read more.


It is a practice to see if any improvement is needed in the method to help you to obtain more reliable and accurate results in your main experiment. The pilot experiment can be used to calculate the following: * The amount of fruit tissue to be used as insufficient amounts could make it difficult to measure the volume of juice thereby giving inaccurate results * The volume of pectinase needed, as if too small an amount is used then reaction time will be very slow and vice versa if too much is used * The range of temperatures to use, as if the temperature is too high the enzyme may denature which is a waste of the resources whilst giving inaccurate results * The time to equilibrate at each temperature before combining the enzyme and the substrate * The time to leave the reactants in the water bath giving enough time for the substrate molecules to turn into product * The type of fruit to use as some may not contain enough pectin therefore producing less juice Apparatus: -3 x 5cm� enzyme -3 x 5g fruit tissue -100 cm� measuring cylinder - Peeler to peel cellulose layer off the fruit -3 test tubes to hold the enzyme and fruit together -Syringe to obtain 10cm� of the Pectinase -Bunsen Burner, tripod, gauze, heat proof mat and large (250cm�) beaker to create the water bath to equilibrate the fruit and enzyme -Cling film to prevent oxidation of fruit and evaporation of enzyme -Filter paper and funnel to filter the juice obtained -100g weight to put on top of the filter paper whilst juice is being extracted -Stop clock to measure the time the mixture has been left -Grater to obtain large surface area of fruit tissue -Glass rod to stir the reactants together -Electronic weighing balance to weigh fruit accurately -Thermometer to ensure temperature is remaining correct -Distilled water to dilute enzyme to a 1:1 concentration -White tile -Safety goggles to protect the eyes -Marker pens ...read more.


In comparison with my pilot experiment the results were quite similar signifying the fact that the results obtained were quite accurate and reliable and they proved my hypothesis correct that the average volume of juice produced would rise as the temperature increased, until after the optimum temperature where denaturing takes place where the rate of reaction decreases again. Evaluation The pilot and main experiment results signify the same effects of pectinase upon pectin. In the preliminary there was insufficient mass of tissue and amount of enzyme. I increased both of these in the main experiment to obtain more accurate and reliable results. In the main experiment I used a range of different temperatures to achieve a smoother and accurate curve on my graph as there were not vast gaps between results allowing us a more calculated prediction of the results in between. These results are more reliable also because I repeated the results for each temperature 3 times. I also increased the time left to filter and the time left in the water bath from 5 minutes to 10 minutes, allowing the maximum volume of juice to be obtained in the revised experiment. I didn't achieve many anomalies however there were some results which were not on the path of the curve and we see an abnormal bend. There could be several reasons for this: Firstly the mass of apple may not be measured correctly. Secondly I maybe did not mange the time efficiently on the stop clocks. Thirdly it could have been the fact that the pectinase solution was not diluted correctly causing a change in concentrations at different temperatures. Perhaps the water bath temperature was not correct leading to slight errors such as the above If I were to repeat the experiment I would use more accurate and stable water baths and accurately measure the concentration of the enzyme. On the whole I was fairly happy with my results and the general curve of the graph is similar to the prediction in the hypothesis. ...read more.

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