|  |
 |
 |
 |
|
|
|
Printable Version
E-mail to a Friend
APA | MLA | |
Enzymes are organic catalysts used to speed up chemical reactions in organisms, without affecting the outcome. A catalyst increases the rate without being used up, which is why they are used in industrial processes were there is a need for high temperatures and pressures.
Enzymes are globular proteins made up of amino acids which are linked together in many different ways due to the complicated three-dimensional shape. A few of the amino acids on the surface of the molecule fold inwards making a specific indentation. This specificity which is achieved is called the
active site. This is the point at which the enzyme comes into contact with the substrate. Therefore specific enzymes will catalyse specific substrate. Once the enzyme and substrate join they form an enzyme-substrate complex making it possible for substrate molecules to be brought together to form an enzyme-product complex.
The product is then released leaving the enzyme free to take part in another reaction. This mechanism is known as the Lock and Key model, meaning the substrate has a complementary shape to the enzymes active site. Another more recent mechanism is that of the induced fit model, where the substrate does not actually fit into the active site(it is not complementary) but the active site is flexible and moulds to fit the substrate.
Both these mechanisms are reversible, as all enzyme catalysed reactions.
A + B ==== C If the concentration is higher on the products side (C) the reaction will go from right to left. If the concentration is higher on the reactants side (A + B) then the reaction will go from left to right.
When a chemical reaction occurs bonds will be broken and formed in the reacting molecules and products. Breaking the bonds in the reacting products requires energy, endergonic, whereas forming bonds in the products gives out energy, exergonic. All reactions start with the breaking of bonds and the energy needed to break them is the activation energy (E ). Enzymes, like all catalysts, work by lowering the activation energy for the reaction they are catalysing. The larger the activation energy the slower the reaction will be, as not all substrate molecules will be able to overcome the activation energy “barrier”. Enzymes reduce the activation energy so that most of the substrate molecules can overcome the activation energy “barrier”.
There are several factors which can affect the activity of enzymes, these are temperature, pH, concentration of enzymes, concentration of substrate and the presence of inhibitors.
Temperature
If the temperature is increased there will be an increase in the kinetic energy of molecules. This will mean increased movement of enzymes and substrate molecules meaning more collisions between them, therefore more conversions of substrate into products. However, after a certain point, the higher temperature begin to denature (change the shape) of the enzyme molecule. The high temperatures begin to break the Hydrogen bonds between the amino acid R-groups of the polypeptide chains within the tertiary structure. This damage to the tertiary structure of the protein means the active site is also denatured, therefore substrate molecules are unable to combine with it, and the reaction rate decreases. The optimum temperature is the temperature where the reaction takes place most rapidly, this is just before the high temperature begins to denature the enzyme. Most enzymes within the human body have an optimum temperature of about 40’C, whereas some bacteria have an optimum temperature of 95’C.
pH
Enzymes normally have an optimum pH at which they function best at, because a change in pH means a change in the H ions affecting the ionisation of R-groups in the amino acids. This affects the active site by denaturing it. Within the human body the optimum pH is 7 normally with a few exceptions. Either side of this optimum would decrease the rate of enzyme-catalysed reactions and any extremes at either side results in the denaturing of the enzyme.
Concentration of Enzymes
Enzymes are able to work over and over again without being used up. They work efficiently at low concentrations. As the substrate molecules are in excess the rate of reaction is limited by the concentration of the enzymes. So if the enzyme concentration is increased the reaction rate will increase but after a certain point there will be too many enzymes and not enough substrate and the reaction rate will level off.
Concentration of Substrate
If the substrate concentration is low then not all the enzymes will be able to react due to the limit of substrate available. The more substrate, the more possibility for enzyme-substrate reactions to take place. After a certain concentration though there will be too many substrate and the enzymes will be working at their maximum capability. This maximum point is called Vmax, which is when all active sites at any moment are occupied with substrate.
Inhibitors
Inhibitors are chemicals which reduce the enzyme-substrate reaction rate, by altering the active site. There are various types of inhibitor. There are reversible and non-reversible inhibitors. Reversible ones bind to the enzyme temporarily; their affect is not permanent, whereas non-reversible inhibitors bind permanently leaving the enzyme denatured and unable to carry out further reactions.
Reversible inhibitor can be competitive or non-competitive. Competitive inhibitor have a molecular shape similar to that of the substrate, so they are able to fit into the active site of he enzyme, preventing the substrate from occupying it; therefore reducing the reaction rate. As this is temporary, when the inhibitor leaves the substrate may occupy the active site once again. The concentration of inhibitor and substrate will affect the degree of inhibition. Non-competitive inhibitors do not become attached to the active site but instead bind to other parts of the enzyme changing the shape of the whole enzyme, including the active site, so that it can no longer bind substrate molecules. They decrease Vmax of the reaction rate. Non-competitive inhibitors can bind fairly weak making them reversible whereas the ones that can bind tightly are non-reversible and leave the enzyme permanently damaged.
• Immobilisation of Enzymes
There are five basic methods of immobilisation.
o Absorption onto a material
o Covalent Bonding
o Cross-linking between enzyme molecules
o Entrapment within the internal structure of a polymer
o Encapsulation within a selectively permeable membrane.
There are several advantages to the immobilisation of enzymes: they are more stable at high temperatures, they are more resistant to change in pH, they are less likely to be degraded by organic solvents, the products are uncontaminated by the enzymes, they can be collected and re-used. These advantages are especially important for industrial processes, which need high temperatures and extremes of pH.
Tryspsin - enzyme
Trypsin is an enzyme secreted into the intestine, where it acts to hydrolyse proteins into smaller peptides or amino acids. This is necessary for the uptake of protein in the food. Trypsin catalyses the hydrolysis of peptide bonds. The enzyme reaction that trypsin catalyses requires a significant activation energy but it thermodynamically favourable. Trypsin is produced in the pancreas in the form of inactive zymogen, trypsinogen. It is then secreted into the small intestine, where the enzyme enterokinase activates it into trypsin by proteolytic cleavage.
Trypsin optimal pH is about 8 and its optimum temperature is about 37’C.
During my experiment I will be using laboratory trypsin.
Casein - substrate
The principal protein fraction of cows' milk. It accounts for about 80% of the protein content and is present in concentrations of 2.5–3.2%. Casein is a mixed complex of phosphoproteins existing in milk as colloidally dispersed micelles 50 to 600 nanometers in diameter. Caseins can be separated from the whey proteins of cows' milk by gel filtration, high-speed centrifugation, acid precipitation at pH 4.3–4.6, and coagulation with rennet and as a coprecipitate with whey proteins.The early production of casein isolates was stimulated by their application in industrial products such as paper, glue, paint, and plastics. These applications have been replaced by petroleum-based polymers. Thus the emphasis has shifted to their utilization in food systems, where they add enhanced nutritional and functional characteristics. They are widely used in the formulation of meat products, coffee whitener, processed cereal products, bakery products, and cheese. The enzyme trypsin can hydrolyze off a phosphate-containing peptone. Trypsin when added to caesin will speed up the time for the suspension to go clear.
Copper(ii) Sulphate - inhibitor
Copper(ii) sulphate is a common salt of copper. Copper sulfate exists as a series of compounds that differ in their degree of hydration. The anhydrous form is a pale green or gray-white powder. The hydrated form is bright blue, which is the kind I shall be using in my experiment. It can be used to plate metals with copper, as a fungicide or herbicide, or as a chemical test for water in which the white anhydrous form turns blue on contact with water. It is used in Benedict's solution to test for reducing sugars, which reduce the soluble blue copper(ii) sulphate to insoluble red copper(i) oxide. Copper(ii) sulphate is also used in the Biuret reagent to test for proteins. Mixed with lime it is called Bordeaux mixture which is used in agriculture. Other uses include hair dyes and the processing of leather and textiles. Copper sulphate is also used to test blood for anemia.
|
|
 |
 |
 |
|
|
|
