Preparation and Assay of Phenolase and Peroxidase

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Title: Preparation and Assay of Phenolase and Peroxidase from Sweet and Irish Potato Aim
To design and conduct an experiment to demonstrate the presence of enzyme activity in the preparation provided. To examine the effect of the inhibitors provided.
To test whether the other phenolic substrates provided can be oxidized by the enzyme preparation. To test for the presence of peroxidase activity in the enzyme preparation. To test the effect of the inhibitor provided on peroxidase activity

alk about enzymes..their structure- tertiary level etc.
(ii) talk about factors that affect enzyme activity...do not just state the factors, actually talk about the effect. eg. heat would affect the bonds of the polypeptide chains that make up the enzyme and cause 3D shape of enzyme to break down and denaturation occurs.. TALK about other factors such and effect of inhibitors (types of inhibition: competitive, non competitive.. etc.) ..ALSO...you used ascorbic acid as the inhibitor...talk about the effects of this inhibitor on the respective enzymes used.. (iii) then, you dealt with two enzymes in the lab, get some information on that...what substrates are those enzymes specific to.... and talk about absolute and relative specificity...state whether the enzymes have absolute or relative specificity.

Theory: An enzyme is a biological catalyst which speeds up a chemical reaction but remains unchanged itself at the end (taylor et. al, 1997). Enzyme molecules are coiled into specific three dimensional shapes with the hydrophilic R group on the outside of the molecule enabling them to be soluble. The active site of an enzyme is normally a cleft to which another molecule can bind to it. The molecule that is acted upon by enzymes, called the substrate (Jones & Taylor , 2000). Enzymes bind to substrate in two chief processes. The first method is the lock and key theory in which the substrates fits exactly into the active site of the enzyme. The second theory is the induced fit theory, the enzyme takes the shape of the substrate thus forming the active site specific to the substrate ( Hames & Hooper 2005).

Diagram 1 Showing The Lock And Key Theory (Eleison.K Lock and Key theory. 21st August 2010

Diagram 2 Showing The Induced Fit Theory (Webster online dictionary http://www.websters-online-dictionary.org/definitions/enzyme?cx=partner-pub-0939450753529744%3Av0qd01-tdlq&cof=FORID%3A9&ie=UTF-8&q=enzyme&sa=Search#922. Induced Fit theory . Enzymes show different degrees of specificity. Two types of enzyme specificity are relative and absolute specificity. In relative specificity the enzymes acts on substrates that are similar in structure and contains the same type of bonds eg, lipase that hydrolyses ester bonds in different triglycerides. In absolute enzyme specificity the enzyme only acts on one substrate eg, maltase which acts on maltose. This specificity is high and the relative specificity is low. The catalytic activity of enzymes is based on the precision of the protein conformation of the enzyme. In particular, this specificity depends upon the shape of a small part of the enzyme molecule where the enzyme actually comes into contact with the substrate molecule, called the active site. This is a small “cleft” in the surface of the enzyme where certain amino acid residues are exposed. These residues contain substituent groups that bind to the substrate and catalyse its chemical transformation. The substrate molecule fits into the active site and interacts with these amino acids by ionic and hydrogen bonding, forming an enzyme-substrate complex. A reaction then occurs, and the product leaves the active site. If an enzyme is broken down into its component amino acids, its catalytic activity is always destroyed. Thus the primary, secondary, tertiary, and quaternary structures of enzymes proteins are essential to their catalytic activity. Factors that affect enzyme activity are the effect of substrate concentration,...
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