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.

Pectin, which is found inside the apple puree I will be testing the pectinase on, can form gels that will bind up liquids. They do this by binding water. However they can only do this when the pectin molecules are very large, for example in fruits such as apples. In fruits pectin naturally functions as a type of 'glue' to help hold the cell wall together. The middle lamella in plant cells, which is between two cells, is densely packed with pectin, indicating that pectin helps hold cells together. This also suggests that digesting pectin by use of enzymes would also make access to the cellulose easier.

this diagram shows the stucture of plant cell, indicating the typical positioning of pectin within the cell - source www.bmc.co.uk

To begin the breakdown to pectin, therefore pectinase must split the bonds between the carbon 1 and the carbon 4 atoms of galacturonic acid, thus shortening the chain into smaller molecules. This type of reaction is known as a catabolic reaction, where large molecules are broken down into smaller ones.

PH is a measure of the number of H+ ions in a substance. There are 3 ways pH can alter enzyme activity. Every enzyme has an optimum pH. This is a pH level that the enzyme works fastest at. For example Peptidase, found in the acidic environment of the stomach has an optimum pH of 2.4, this is a highly acidic pH.

However, if the pH were to change from the optimum level then it would affect the charge on the active site of the enzyme, or the charge on the substrate molecules, in this experiment on the apple puree. Changes in charge on the active site or substrate molecules will slow down the rate at which the enzyme can form the enzyme-substrate complex. The diagram below shows how different pH can alter enzyme activity:

The second way that pH can alter enzyme activity is by changing the properties of the amino acids in the polypeptide chain that builds up each enzyme. Enzymes are just special globular proteins. If we were to look back at the secondary structure of globular proteins we could see that hydrogen bonding is important in the formation of proteins.
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Amino acids, that build up proteins, contain both a -CO (carboxyl group) and an -NH (amino group). These groups are charged, -CO is slightly negative and -NH slightly positive, so they attract each other and allow for bonds between amino acids to occur. If pH were to change the charge on one of these groups then where the groups would previously attract each other they would now repel, and this would change the properties of the entire globular protein. Also because the -NH group is a base, it is able to pick up H+ ions, this could change ...

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*** 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. Planning 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. Implementing 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 Communication 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