E + S ES where E = Enzyme, S = Substrate(s)
ES = Enzyme/substrate complex and P = Product(s)
ES E + P
The enzyme is unchanged by the reaction.
Cellulase catalyses the breakdown of cellulose to β-glucose sub-units (or sugar acids)
cellulase
Cellulose β-glucose
Plant cells have cell walls which give structural rigidity and strength to the cell and protect them from physical damage.
These cell walls are composed of cellulose, hemi-celluloses, pectins and proteins with secondary cell walls containing less pectins than primary cell walls. The middle lamella is primarily composed of pectins. Cellulose is a polysaccharide made up of β-glucose sub-units connected by condensation reactions to form β 1,4 glycosidic bonds. Alternate glucose molecules are “flipped over” (see diagram) so that cellulose consists of long straight chains.
The chains are further linked by hydrogen bonds to form microfibrils which are in turn linked to form fibres of cellulose. These have a very high tensile strength and are insoluble and so resist hydrolysis. These characteristics make cellulose an efficient structural molecule. Pectins and hemi-celluloses are also polysaccharides. Pectins are composed of galactose and galacturonic acid residues whereas hemi-celluloses are composed of pentose sugars and sugar acid residues. Cellulase does not act on either of these groups.
Fruit juice is formed from the fruit cell sap. In order to extract it, the cell plasma membrane must be breached. Since one of the cell wall’s functions is to protect the plasma membrane from damage, destruction of the cell wall would be expected to increase cell sap loss. Cellulase performs this function by catalysing the breakdown of the cellulose in the cell wall allowing greater escape of sap as fruit juice. The graph shows that increasing the enzyme concentration generally results in greater production of fruit juice as more cellulose is broken down. The 0% cellulose result confirms that cell wall breakdown is necessary for release of cell sap.
Enzyme theory predicts that the reaction rate increases with increasing substrate availability (to a maximum) and increasing enzyme availability. Increased concentration of enzyme provides more active sites for substrate binding. So long as excess substrate is available, the reaction rate increases in proportion to enzyme concentration (see over).
If substrate is limited, an increase in enzyme availability does not result in increased reaction rate because active sites have no substrate to bind and the reaction rate plateaus.
The experimental graph shows a tendency towards a plateau of the reaction rate with increasing enzyme concentration indicating that substrate concentration (i.e. apple cell wall cellulose) was limiting. However, a plateau was not actually reached as high enough enzyme concentrations were not used (see D 1 g below).
- EVALUATION
- Limitations of the Experimental Method
-
Replicates
No replicates were carried out. At least 3 sets of data should have been obtained in order to be certain that results are meaningful, to identify the trends and to identify anomalous results.
-
Even Puree?
The apple pulp could not be evenly pureed with the blender because the volume was too small. There were some lumps. This could have resulted in variation in the total surface area of apple presented to the different enzyme solutions and therefore in cellulose available for reaction. A blender designed for smaller quantities should have been used to give greater consistency. It is also possible that different parts of the apple contain different amounts of cell sap. Uneven blending and mixing would contribute to inaccuracy.
-
Equal division of puree
Although done as accurately as possible, exact equal division by weight of the puree was difficult because of the lumpy consistency. This would also have resulted in a variation in available cellulose.
-
Accurate measurement of volume
The total volume produced was measured by pipetting out of the collection flask. Since some solution was left in the flask, this was not very accurate. Pouring the liquid into a measuring cylinder would have allowed more accurate measurement. (In practice, this limitation of the method was probably not significant as a source of inaccuracy).
-
Immersion of apple
The total volume of apple produced was such that it was not initially all immersed in the 10cm3 of enzyme solution. Thus, not all exposed cell walls would have been accessible to the cellulose. 15cm3 should have been used for each sample.
-
Time allowed for experiment
By the Q10 effect, an increase in temperature of 10oC gives an increase of 2 to 3 times in the reaction rate of enzyme catalysed biological reactions. Thus, assuming a room temperature of 20oC, immersion at 40oC would have increased the reaction rate by 4 to 6 times. However, since the apple was only immersed for one and a half hours, this equates to about seven hours at room temperature compared with the recommended twenty-four hours. It is possible that a different pattern would have emerged once the equilibrium point had been reached.
-
Range of enzyme concentration
A wider range should have been used to establish whether and at what point a plateau was reached.
-
Anomalous Result
The volume produced with 1.5% cellulase showed a very small net decrease compared with the baseline. Volume measurement may have been inaccurate (see point d). Alternatively, this sample may have been composed of less well pureed apple (see point b) resulting in a lower surface area of apple cell wall for cellulase action. Mistaken use of 0% cellulose instead of the 1.5% solution would also explain the anomaly. However, since no replicates were done (point a) it is not strictly valid to assume that this was a result of experimental error. Replicates would establish whether or not the volume produced with 1.5% cellulose confirmed the trend towards a plateau shown by the other results. If the 1.5% cellulase datum point had been in line with the others, a net volume of about 6.2 cm3 would be predicted.
- Extensions to the experiment
-
Effect of temperature and pH
The experiment could be repeated using a range of immersion temperatures from 10oC to 90oC and a range of pH values to establish the optimum temperature and pH for cellulase activity.
An enzyme’s activity depends on the precise conformation of its active site (both shape and charges) which in turn depends on the shape of the entire protein (tertiary and quaternary structures). An enzyme which has a structure changed by external factors is said to be denatured. Ionic and hydrogen bonds can be broken easily by changes in pH and by temperature changes as they are weak or fairly weak. Such denaturation is reversible as the bonds can easily reform. In fact enzyme function depends on such changes. Covalent bonds require higher temperatures to be broken chemically and such denaturation is irreversible.
Heating a reaction solution has two conflicting effects (see graph). Kinetic energy of enzyme and substrate both increase allowing greater probability of collision and interaction. However, above a certain temperature this greater reactivity is offset by increased denaturation giving an upturned U-shaped graph for reaction rate against temperature. The maximum reaction rate observed is the optimum for that particular enzyme
-
Effect of Pectinase
Since the plant cell wall is also composed of pectins which are broken down by pectinase, the experiment could be repeated using pectinase and also a combination of cellulose and pectinase to establish whether pectinase is more efficient at breaking down the cell wall than cellulose and whether both enzymes together have a greater effect than either alone (as would be predicted). Pectins bind water tightly and therefore prevent loss of cell sap.
-
Varying sources of substrate
Different varieties of apple could be used using the same weight and consistency of puree for each experiment. The effect of storage and age of the fruit could also be investigated.
-
Rate of extraction of juice
This could be established by immobilising the cellulase - for instance in alginate beads mixed with the substrate in a column with an outlet at the bottom – and measuring the liquid produced per unit time under different conditions. The initial reaction rate could be measured by plotting volume produced against time and measuring the gradient of the asymptote at zero time. Initial reaction rates at different substrate concentrations could be plotted against substrate concentration to investigate Michaelis-Menten kinetics of the enzyme. A Lineweaver Burk plot of the reciprocal of initial velocity against the reciprocal of substrate concentration would give the Michaelis constant which gives an indication of the turnover rate of the enzyme.
-
Conditions for optimum fruit juice extraction
The experiment could be repeated with differing incubation times at the optimum temperature and pH (see a) above) to find the conditions under which maximum fruit juice extraction occurs. This would be the pilot stage of a design to produce apple juice commercially.
-
REFERENCES
BOOKS
Clegg CJ and MacKean DG Advanced Biology Principles and Applications, 2nd edition, 2000, John Murray (Publishers) Ltd, (London)
Pickering WR AS and A Level Biology through diagrams, 2002, Oxford University Press (Oxford)
Indge B, Rowland M, Baker M A New Introduction to Biology, 2000, Hodder & Stoughton (London)
INTERNET SITES
ncbe.reading.ac.uk/NCBE/PROTOCOLS/INAJAM/PDF/JAM01.pdf