The objective of the assessed practical is to investigate the effect of different concentrations of sodium chloride on the rate of hydrolysis of starch using the enzyme diastase, or more commonly known as amylase. Therefore, the principal behind the investigation is to look into different factors that affect the rate of enzyme activity.Background InformationStarchStarch is made up of combinations of two different polymers of alpha glucose, amylose and amylopectin. Amylose is composed of approximately 300 glucose units connected by alpha 1,4 glycosidic bonds. Amylopectin is a branched chain composed of approximately 1500 glucose subunits, which are connected by the cross linking of alpha 1,4 glycosidic bond chains by alpha 1,6 glycosidic bonds.
The chains branching off the CH2OH on alpha glucose molecules cause the polysaccharide to take up a helical structure. The diagrams below represent the points stated:Amylose - alpha glucose molecules connected by alpha 1,4 glycosidic linksAmylopectin - branched chain of alpha glucose molecules joined by 1,4 and 1,6 glycosidic cross linksFrom the diagrams above it is apparent that there are very few free ends on a starch molecule as a whole, this would therefore simply mean that there are not many regions on the molecule where hydrolysis by amylase could commence.An easy method of testing whether starch is present is to add 2cm3 of the solution that is to be tested into a test tube. Add 2 drops of the solution into a spotting tile using a pipette. Add a drop of iodine solution on to the same spot on the tile.
If starch is present the solution should change in colour to blue-black. The principle behind this experiment is due to the ability of iodine to bind to the centre of the starch helix, which forms a starch-iodine complex. This complex is blue-black in colour; explaining the colour change in a solution containing starch after iodine has been added.'In flours and semolinas, starch is quantitatively the main component (on average around 60-65%). Chemically it is a glucidic substance, composed of microscopic grains of diameter between 2 and 100 thousandths of a millimetre, comprising approximately 25% amylose (a linear chain of glucose units) and 75% amylopectin (chain with a branched structure of glucose units).
These two components of starch are physically combined in a crystalline kind of reticular structure. In plain words, starch is a compound of sugars, grouped together in its two main fractions, amylose and amylopectin. Starch is insoluble in water, while its two essential fractions have differentiated behaviours: amylose is soluble in hot water and amylopectin is not.The extract of text was taken from www.
professionalpasta.it, as it provides information on starch and its behaviour, it also gives an idea of what kind of substances it is present in.EnzymesEnzymes are substances that act as catalysts. They reduce the activation energy required for a chemical reaction to take place and therefore speed up the overall rate of reaction without changing the temperature at which the reaction occurs. Enzymes are biological catalysts, with the ability to increase the rate of reaction by a factor of at least a million. Without the presence of enzymes many reactions in the cell would be too slow to sustain life and many reactions would require very extreme conditions in order to occur; most cells would be destroyed.
Most enzymes are globular proteins made up of folded peptide chains. (However, it has recently been discovered that substances other than proteins have catalytic properties too). The irregular bending and folding of the polypeptide chains into a compact globular shape is referred to as the tertiary structure. Types of bonding found in the tertiary structure of proteins include, ionic bonding, hydrogen bonds and disulphide bonds. The tertiary structure is important to enzymes, as the maintenance of the shape of the active site within an enzyme is vital for its activity.Enzymes are not destroyed or used up by the reactions they catalyse and there presence does not alter the end products, however, they do wear out and have to be constantly replaced.
Catalysed reactions are reversible and the enzymes are highly specific to a particular reaction. The mechanisms of enzyme action can be explained using a simple theory called the lock and key hypothesis.Factors, which affect the action of enzymes, include:1. Temperature - enzymes are inactive at temperatures lower than the optimum, and denatured at temperatures above the optimum temperature.2.
PH - enzymes are denatured at extremes of pH, and all enzymes have an individual optimum pH.3. Substrate concentration - activity of enzymes increases with increasing substrate to a maximum, where it then levels off due to saturation of the active sites.4. Enzyme concentration - this increases the activity of enzymes continuously, provided that substrate concentrations are maintained at a high level.
5. Enzyme inhibition - enzyme inhibitors reduce the rate of an enzyme-controlled reaction.6. Cofactors - some enzymes can only function efficiently in the presence of other substances.
CofactorsCofactors must be further investigated in the background information, as sodium chloride is a possible cofactor for the reaction between starch and amylase. Some enzymes only work in the presence of another chemical which serves as a 'helper,' such enzymes are called cofactors.Sometimes a cofactor can be a metal ion such as (Zn2+), which would encourage the binding of the substrate to the enzyme or it may have an active role in the reaction in centre of the enzyme active site.However, other times a cofactor can be a complex non-protein organic molecule also known as a coenzyme.
They usually act as carriers, carrying chemical groups of atoms from one active site of one particular enzyme to the active site of another enzyme. Coenzymes that are tightly bound to enzymes are called prosthetic groups. They carry out the same function as coenzymes and can be thought of as 'built in' coenzymes.In relation to NaCl it can be suggested that in the solution the (Na+) and (Cl-) ions are allowed to move around freely and individually, therefore (Na+) being a metal ion could be the cofactor encouraging the binding of the substrate to the enzyme or it may have an active role in the reaction in the active site of the enzyme.InhibitionAlthough unlikely, the Na+ ions or the Cl- ions could in fact inhibit the enzyme from functioning properly.
Therefore it would be appropriate to collect some background information, this will help when it comes to producing a hypothesis.Competitive InhibitionThis functions through the competition between the inhibitor and the substrate for the active centre. When the inhibitor molecules bind to the enzymes, the active site is plugged up or the process of substrate entry is slowed down.Non-competitive InhibitionIt occurs when the inhibitor, which does not have to be similar to the natural substrate in terms of structure, binds to the enzyme in a location other than at the active site.
This denatures the active site becoming unable to break down the substrate molecules.This can either be reversible, where by the inhibitor eventually leaves and the active site is restored, or it could be irreversible. Irreversible inhibition occurs when the inhibitor either permanently attaches to or heavily denatures the enzyme, resulting in the tertiary structure becoming unrestorable.An example of permanent non-competitive inhibition is nerve gas, which block nerve message transmissions, resulting in death. Another example would be penicillin, which permanently blocks the pathways that certain harmful bacteria use to accumulate their cell wall components.
AmylaseMany organisms can digest starch therefore; amylase must be commonly manufactured in nature. For example, human saliva and pancreatic secretions are composed of large amounts of amylase for starch digestion. The specific bond 'attacked' by amylase is dependent on the sources of the enzyme. Based on the points of attack in the glucose polymer chain, the enzymes can be classified into two categories, liquefying and saccharifying.Amylase, that randomly attacks only the alpha 1,4 glycosidic bonds belongs to the liquefying category.
The hydrolysis reaction catalysed by this class of enzyme is usually carried out only to the extent that, for example, the starch is turned into a soluble enough substance to allow easy removal from starch-sized fabrics in the textile industry. The paper industry also uses liquefying amylases on the starch used in paper coating where breakage into the smallest glucose subunits is actually undesirable.On the other hand, the fungal amylase belongs to the saccharifying category and attacks the second linkage from the non-reducing terminals of the straight segment of the starch molecule. This results in the two glucose units splitting off at a time. The product is a disaccharide called maltose.
The bond breakage is evidently more widespread in saccharifying enzymes than in liquefying enzymes. The starch chains are chopped into smaller bits pieces.Glucoamylase, which can be a component of amylase, selectively attacks the last bond on the non-reducing terminals. It can act on both the alpha 1,4 and the alpha 1,6 glycosidic linkages, which therefore results in simple glucose units splitting off into the solution. Fungal amylase and glucoamylase can be used together to convert starch to simple sugars.
The practical applications of this type of enzyme mixture include the production of corn syrup and the conversion of cereal mashes to sugars in brewing.HypothesisThe above shows the role of the enzyme amylase and the substrate starch in the following experiment. The enzyme breaks down the starch molecules into dextrin, maltotriose, maltose, and glucose molecules. The methods of breaking down the starch are explained in the plan.In relation to the information about this topic in the sections above it would be appropriate to analyze the information to produce an educated hypothesis on how the substances involved will behave.
The basis of the experiment is to investigate the effect of different concentrations of NaCl on the rate of hydrolysis of starch using amylase, therefore it would be appropriate to comment on the sodium chloride first.Based on the information collected it is apparent that the Na+ ions in the NaCl solution are cofactors, which are described in the sections above. This is because Na+ ions are metal ions and therefore more likely to be cofactors than inhibitors, also they are more likely to be 'helpers' in terms of binding rather involved in the reaction themselves. This is because the reaction can take place without Na+ ions as well, if the Na+ ions were involved in the break down of the starch this could not happen.
According to this the effect of increasing the concentration of NaCl would be an overall increase in rate of reaction. This is because the more Na+ ions, the more binding of the substrate to the enzyme active site, or the more active the Na+ ions are in the break up of starch.The greater the concentration, the more Na+ ions are in the solution. More Na+ ions in solution means that more substrate-enzyme complexes are helped to be formed. The more the chances there are of enzyme-substrate complexes forming, the greater number of starch molecules get broken down.
If more enzyme-substrate complexes are forming, the rate of reaction increases.This is known as the collision theory. Therefore, it can be concluded that the rate of an enzyme catalysed reaction increases with increasing concentration of necessary cofactors, provided that the other conditions such as pH and temperature, are kept at a constant.I predict that if an increasing concentration of NaCl is used with no change in volume of enzyme used, a saturation point may be reached.
This is where the rate of reaction reaches a maximum and then it levels off due to saturation of enzyme active sites. At this point the active sites of enzymes are occupied with substrate molecules and any extra substrate molecules have to wait for a vacant active site. The presence of more Na+ ions will speed up the binding of substrates to the active sites, however, there will only be so many enzymes free for binding at any point in time.
