out how different concentrations of the enzyme pectinase affect the degradation of the substrate pectin

PLANNING: -

Aim:

My aim is to find out how different concentrations of the enzyme pectinase affect the degradation of the substrate pectin, and hence the volume of clarified apple juice produced in a specific duration of time.

Hypothesis:

Will different enzyme concentrations have different effects on the yield of clarified apple juice obtained from apple pulp of equal masses, in a given time?

Biological Knowledge:

Enzymes are a class of proteins, which are biological catalysts responsible for speeding up and controlling metabolic reactions, without their shape being altered by the reaction. Pectinase (the enzyme I will be using for my investigation) is under the class of enzymes named hydrolases, which are responsible for catalyzing reactions between a substrate and water, and bind water to certain molecules. In this way, larger molecules are broken up into smaller units. This class of enzymes catalyses the cleavages of peptide bonds in proteins, glycosidic bonds in carbohydrates, and ester bonds in lipids. Enzymes are specifically designed for their target molecules, their substrates, by virtue of their shape, size, and the chemical charges that precisely fit and bond to the substrate molecule. The section of the enzyme that binds the substrate is called the active site. An advantage of using enzymes is that they are specific and do not interact with other components in the food or beverage. Another advantage is that by catalyzing the same reactions repeatedly as long as the substrate is available (referred to as turnover), they can be used at very low concentration. Eventually, as the end products of the enzyme reaction increase in concentration, the reaction is temporarily inhibited by feedback inhibition.

This shows how the catalysis of pectin by the enzyme pectinase occurs:

The cell wall of fruit comprises a complex mixture of various polysaccharides and some protein. Before fruit juice can be extracted from fruit, the cell wall has to be ruptured. This can be achieved by physically macerating the fruit or this can be accomplished by the use of enzymes. The cell wall contains cellulose, hemicelluloses, pectins and proteins. Pectinase specifically targets pectin, which bonds the long cellulose strands together, in the middle lamella, to form the outer core of the apple. Pectins are large polysaccharide molecules, made up (mainly) of chains of several hundred galacturonic acid residues. Therefore, because fruit cell walls have such a complex structure of interwoven polymers, no single enzyme will break down cell walls. Complete liquefaction of a cell wall is likely to require a complex cocktail of carbohydrase enzymes. In fact, there is no single enzymes that will completely break down complex pectins. Commercial ‘pectinases’ are usually mixtures of enzymes such as polygalacturonases and pectin lyases, which result in the break down of pectins. Breaking down these bonds would mean the cellulose strand could move freely and therefore form a liquid. Pectinases are obtained commercially from fungi, particularly species of Aspergillus and Penicillium. It is the bacteria and other fungal pectinases that help in the soft rot of fruits and vegetables, contributing to spoilage and decay. Hence pectinases break down the cell wall of plants (with no wood) this way, they are widely used in the fruit juice industry, where they are widely used to help extract, clarify and modify fruit juices. The optimal activity of these enzymes is at a pH of 5.5 and generally below 50°C, around 35°C.

Diagram of a Plant Cell Wall: Model Of Pectin Network In Plant Cell Wall:

Commercial Uses Of Pectinase:

Clarification of juices/wines.
Aiding the disintegration of fruit pulps.
By reducing the large pectin molecules into smaller units, and eventually into galacturonic acid (compound become water soluble) loses their suspending powder.
Viscosity is reduced.
Insoluble pulp particles rapidly settle out.

Factors That Affect The Enzyme Pectinase:

Since pectinases are enzymes the same factors that affect enzymes also affect them. These are:

Fluctuations in temperature will affect the activity of the pectinase. As temperature increases, the kinetic energy of the pectin and pectinase molecules increases and this results in an increase in movement. The faster these molecules move, the more frequently they will collide with each other, increasing the number of enzyme-substrate complexes formed and therefore increasing the rate of reaction. An increased rate of reaction would result in more pectin being catalysed in a given time, so leading to an increased rate of juice flow. Although highly unlikely in this experiment, significant increases in temperature would cause the atoms within the pectinase molecules to vibrate. This breaks the hydrogen bonds holding the molecules in their shape and so altering the three-dimensional shape of the pectinase molecules to such an extent that their active sites no longer fit the pectin molecules. This would denature the pectinase and result in the lowering of the rate of juice flow.

Variations in the pH will affect the activity of pectinase. Just like enzymes have an optimum temperature they also have an optimum pH. However, this value varies from enzyme to enzyme. For example in the stomach where the conditions are acidic the optimum pH level will be at a low pH value (pH2). In the case of pectinase, which I will be experimenting on, the enzymes work best at a ph of 5.5. At low pH levels the enzyme becomes inactive; however this is a reversible situation since when the enzyme restores its optimum pH, the normal activity of the enzyme will resume. The result of an extreme pH, however, may cause an irreversible change to the enzyme. It may cause the enzyme to change: its protein structure, the binding of the substrate to the enzyme, the properties of amino acids or co-factors involved in the catalytic activity of the enzyme and the ionisation of the substrate.

The substrate concentration. Increasing the concentration of pectin, will increase the rate of reaction, providing not all active sites are in use, because the more pectin molecules there are in the solution, the more enzyme substrate complexes form. This will cause increased masses of apple juice and so increased flow rates to be obtained, as an increase in the rate of reaction will result in more pectin molecules to be catalysed. This remains true up to a point where all active sites are occupied and the volume of pectinase becomes the limiting factor causing no further increases in the rate of reaction.

Enzyme inhibition. If there are more inhibitors, competitive or non-competitive, compared to the substrate concentration this will reduce the reaction rate in which juice flows. This is because competitive inhibitors compete for the active sites hence leaving less active sites for the pectins (the substrates) to get attached to and be catalysed by the pectinases. However as the substrate concentration increases, less inhibition will occur since there are more substrates meaning the substrates have a greater chance of finding a free active site, leaving fewer to be occupied by inhibitors. Competitive inhibition is reversible, unlike non-competitive inhibition, where the structure of the enzyme is being denatured and hence having a very low reaction rate in which juice is produced.

The enzyme concentration, which will be the factor that I will be testing in my investigation. As the enzyme concentration increases, the rate of reaction also increases. This is because there will be more active sites that can catalyze substrates; thus causing an increase in the rate of activity. Therefore the enzyme concentration and the rate of reaction have a directly proportional relationship. As the concentration of pectinase increases, the number of active sites will also increase. Provided that there is an excess of substrate molecules, the rate of reaction increases in proportion to the concentration of the enzyme, thus resulting in a higher yield of apple juice passing through the measuring cylinder. However if the enzyme concentration is very high in relation to the substrate concentration, there would not be any further increase in rate and it would remain constant. This is because even though there would be many active sites, there would not be enough substrates for catalysis.

Based upon all this biological knowledge I have attained, I want to find out how different pectinase concentration will affect the break down of the substrate pectin in the cell wall of equal masses of apple pulp, and how much clarified apple juice is produced in each pectinase concentration, in a given time.

I believe that as the concentration of pectinase increases so will with the yield of apple juice produced until a point is reached where the pectin concentration and the pectinase concentration equal each other and no extra pectins will be degraded hence no extra ...