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The Effect of pH on Pectinase

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The Effect of pH on Pectinase Hypothesis I plan to investigate the effects of varying pH on the enzyme 'Pectinase'. I will test the yield of apple juice from a set amount of pureed apple at different pH. I think the optimum pH for pectinase is likely to be around pH 5, an acidic pH, as many commercial pectinases are produced from fungi. Before I look at the structure of pectinase, first it would seem sensible to look at the substrate that pectinase will be working on, Pectin. Starch and Pectin are very similar molecules. Starch and Pectin are both polysaccharides. However in Pectin the repeating unit is not glucose as in starch, but galacturonic acid. Galacturonic acid is very similar to glucose, except that the carbon with CH2-OH attached in glucose is replaced by a -COOH in galacturonic acid (shown below). Bonds between carbon 1 of the first galacturonic acid and carbon 4 of the next hold pectin chains together. This sequence continues down the chain. Enzymes are biological catalysts; they alter the speed of reactions without being reactants themselves. There are two theories on how enzymes work. The first is the 'lock and key theory'. This theory suggests that substrate molecule fits perfectly into its specific enzymes active site, and only that enzyme. This can be seen below: The second theory is the 'induced fit theory'. This suggests that the active site on the specific enzyme changes its shape to accept the substrate molecule. The diagram below shows this: Pectinase is an example of an enzyme. Pectinase is often formed from more than one enzyme - polygalaturonase, pectinmethylesterase and pectin lysase are normally mixed to form pectinase. These three enzymes can assist the breakdown and modification of pectins from many plant materials. In industry they are used in the processing of grapes, apples and oranges (a citrus fruit). Commercial pectinase may contain cellulase and other cell wall degraders. ...read more.


The second variable that must be controlled is the concentration of the enzyme pectinase that will be added to the substrate, apple puree. If you increase the number of enzymes in a solution you increase the number of active sites available to substrate molecules. This means that the reaction could take place faster. It is obvious that this variable needs to be controlled; therefore on in each beaker I will place 1g of pectinase. This means the same amount of pectinase is available to each 100cm? of substrate apple puree. Finally the substrate concentration must be controlled. If there is a small amount of substrate, the reaction will take place slowly because not all the active sites are in use. However as substrate concentration increases, so does the rate of reaction, until a point is met where all the active sites are in use and cannot work any faster. By using 100cm? of apple puree in all 6 beakers I can be sure that the substrate concentration is being controlled. Risk assessment In any experiment there are always hazards. When using pH one must be careful not to get any of the pH solution on their hands. This is due to low pH (i.e. pH3) being acidic and higher pH (i.e. pH10) being alkaline. Acids and Alkalis are harmful to human skin because When using a water bath there is the risk of scalding. In my experiment I need to boil the beakers containing apple puree and pectinase, along with pH3, 4, 5, 6, 7 or distilled water, to stop the reaction. I must be careful not to scald myself, and take care whilst also being precise with my measurements. The well being of others must be taken into account so that I do not inadvertently scald other students whilst conducting this experiment. Pectinase is an enzyme and some students may be allergic to some enzymes, they can produce skin rashes or affect eczema. ...read more.


The results I took proved that extreme pH does affect enzyme activity and that the enzyme Pectinase does have a specific pH at which it prefers to work. From my results I was able to discover this pH to be the buffer solution set at pH5. To further extend research into the effects of pH on enzymes there are many possible experiments. The breakdown of Pectin by Pectinase is an example of a catabolic reaction because a large molecule is broken down into its smaller subunits. This means that during the experiment there was still time for some of the pectin to be broken down before the pectinase was irreversibly denatured. However research into enzymes on the effect anabolic reactions, where small molecules come together to form larger ones would provide interesting results. Research into the optimum temperate for an enzyme to operate, would give those in the biotechnologies industry the ideal information for the use of enzymes in for example washing up liquid. It would be very simple to modify the experiment I conducted that looked at the effects of pH, to look at the effects of temperature. If a range of water baths from 20 C to 80 C were used and pH, substrate concentration and enzyme concentration were controlled properly then I could have modified my experiment to look at the optimum operating temperature for pectinase. If chemists can discover the optimum temperature and pH for every enzyme in living organisms then it would give a great insight into reactions within the environment around us. Currently scientists debate whether the induced fit theory, or the lock and key theory provides the answers to how enzymes work. No one knows exactly which of the two is correct but currently research is taking place into which of the two theories most accurately describes how enzymes operate and interact with their substrate molecules. ...read more.

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Here's what a teacher thought of this essay

3 star(s)

*** A well structured report which covers the basic criteria but is lacking depth in key sections.
Research and rationale
There is some background material referring to the action of pectinase but it is a little limited. The student should have included some references to provide a background for the project. Pectinase is used extensively industrially so there is a lot of additional material available.
A testable hypothesis has been formulated and stated concisely. The apparatus is suitable for the project but had not been justified. Insufficient thought had been given to the key variables. The range of pH values was not really adequate. For an A level experiment a minimum of five values of the independent variable is considered to be acceptable. The control would not work as expected since the apple puree must have a pH level which would allow the pectinase to work. The risk assessment is minimal and mentions procedures that were not in the stated method.
The apparatus seemed to have been used competently but the data should have been recorded in a more suitably headed table.
Analysing and Evaluation
A summary table was presented but no graph was included. No statistical analysis of data was included e.g. calculation of standard deviation. The explanations were sound and related to basic biological knowledge. The analysis was not helped by the small range of data obtained which meant the candidate tried to "fill in the gaps".
There was no evaluation of the method or results obtained
The layout was acceptable with suitable headings and subheadings. The data could have been presented more clearly in tables that follow scientific conventions. Spelling, punctuation and grammar were reasonable and scientific terms used correctly.
No background reference were used

Marked by teacher Stevie Fleming 01/01/1970

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