Enzymes are sometimes referred to as biochemistry workhorses because they are utilized by many scientists to speed up a number of chemical reactions. Enzymes are proteins that speed up, or in other words catalyze, specific biological and chemical reactions by increasing the rates of reaction by more than a million factors. An example of these enzymes is Pectinase, which is the enzyme that catalyzes pectin breakdown. Pectin is a part of the cell wall of fruits like tomatoes, mangoes, oranges and apples. Pectins are large colloidal molecules and they are responsible for holding or determining the amount of dispersed particles in extracted juices. Pectins also determine the viscosity of an extracted fruit juice in addition to determining a juice’s suspended particles (Novo Laboratories 34-56).
Pectinase releases juices from within the fruits cells by breaking down, enzymatically, its cell wall. Polygalacturonase is one kind of Pectinase that has been widely studied on, and as a result it is widely used. Its usefulness is because of the jelly like nature of pectin, something that is useful in cementing cells in plants together. Pectinase enzymes are therefore very useful in any process that requires the degradation of plant components (Buchanan, Gruissem and Jones 34). Pectinase enzymes are commonly utilized in speeding the processes of extracting juice from fruits like apples. The enzyme has also been widely used in wine production as it speeds up the extraction of flavors from the grape fruits that have been broken down, in addition to clearing up the resulting juices. Pectinase enzymes can be produced or extracted from Aspergillus niger, which is a type of fungi. This fungus produces Pectinase to aid it in breaking down a plants middle lamella for the purposes of obtaining nutrients, and also for inserting its hyphae (Novo Laboratories 90- 123).
Pectinase can be denatured, through unfolding of the protein, something that makes it inactive as unfolding makes it difficult for the enzyme to connect with its substrate at its active site. The effects of Pectinases can be increased or enhanced by adding a chelating agent to the plant extract or the substrate, and also by pre- treating the enzyme’s substrate with acid. Just like any other enzyme, Pectinases needs a particular optimum environment at which they act best. The enzymes for example, have an optimum pH as well as temperature at which their activity is maximal. A commercial Pectinase like Polygalacturonase for example, may typically be activated or most active at temperatures between 55 degrees Celsius and 45 degrees Celsius, and at a pH of 5.5 to 4.5(Novo Laboratories 67-89).
Objective of the Experiment
Pectinases are used to hydrolyze Pectins, or in other words, to break them down. This catalytic activity of Pectinase results to an increased softening of the fruit. Pectinases are therefore, used to hasten the process of ripening of a fruit, to extract juices from fruits as well as clear the ‘cloudy’ appearance of the extracted fruit juices. The objective of this experiment was therefore, to measure the effects of substrate concentration on the activity of the Pectinase enzyme. The experiment was aimed at finding in what ways the concentration of Pectinase enzyme affected the hydrolysis or breakdown of pectin.
TASK 1
Material and Procedure
The materials of this task included a solution of pectin, a syringe with its plunger removed with a capacity of 10cm3, a stop watch, a clamp stand, a small measuring cylinder in addition to a small beaker. The pectin solution was poured into the syringe with the thumb kept on the nozzle. The solution was poured into a beaker when the thumb was removed and the time it took for the solution to pour into the beaker was measured. The two steps were repeated five times. The pectin solution for each measurement was re- used.
Question 1: The importance of taking several measurements in the experiment
When the appropriate instruments and materials are available during an experiment, and when the researcher has the ability to take and read measurements, an experiment requires no more than just a careful and accurate reading of the scale on the equipment being used. However, most common scientific experiments require and utilize complicated techniques and elaborate equipment. As a result a scientist might need to perform lengthy sequence of operations. These operations sometimes might lead to an improper and inaccurate reading of scale (Youde 7). This is because most measurements are prone to numerous error sources some of which may affect the measurement profoundly by either making the measurements too small or too large. It is therefore the responsibility of the researcher to make sure that these sources are as few as possible. One of the many good ways of reducing and minimizing errors or of enhancing the accuracy of a measurement in an experiment is by taking several measurements, after which these measurement’s average is computed. In other words, by taking numerous measurements, an experimenter ensures that his results are near to accurate.
Question 2: Precautions that must be taken when using syringes to ensure the reliability of the reading
Several precautions must be taken when using various types of equipment in the lab. These precautions help by ensuring that the experimenter attains the most reliable results as possible. For example, when using a syringe in the lab, an experimenter must observe several precautions such as reading the meniscus at the level of the eye, placing a white paper behind the syringe while taking the reading so that the experimenter can be able to clearly see the meniscus level of the syringe. Another precaution that one can observe during an experiment whereby syringes are used is to use a clamp stand when a solution is being drained into a beaker from the syringe. This helps in keeping the syringe at an angle that is vertical. This angle helps the experimenter to attain the most reliable measurements as possible of the time a solution takes to drain into a beaker. Other miscellaneous precautions one can observe include choosing the same end point for each repeat of the experiment to stop the stop watch when timing the time a certain volume of a solution takes to flow into a beaker from a syringe, in addition to repeating the experiment several times.
Question 3a: calculation of the mean time taken for 10cm3 of pectin solution to flow from the syringe into a beaker
C1V1= C2V2
C1= 100%, V1= 10.5, C2=? V2=10
1 x 10.5 = 10 x (?) = 10.5/ 10
=1.05
Mean time taken for pectin to flow into a beaker from a syringe
34.52 + 33.60 + 32.21 + 28.44 + 28.26
=157.03/5
=31.41seconds
Question 3b: the mean rate of flow of the pectin
10.5 + 9.5 + 10 + 10 + 10
=50/5
=10
10/ 31.41
0.31cm3s-1
Question 4a: How would measuring the time taken, by a larger volume of pectin to drain be affected by using a larger syringe
A larger syringe provides a larger surface area for the pectin solution to cling on. This can greatly affect the flow rate of the solution. Also the larger syringe provides a larger volume of air to fill on the unoccupied space. This would affect the flow rate by increasing it as the larger amount of air would be pushing the solution with a greater force in a larger syringe than in a smaller syringe.
Question 4b: Using a digital timer that measures 0.0001 seconds
Just as well, using a digital timer that measures a figure of 0.0001 seconds can bring about unreliable and inaccurate results. This is because 0.0001 is a very small unit that in many cases cannot be measured or accounted for. The difficulties brought about by the inability to account for such a small unit of measurement can bring about inaccurate results which are in turn unreliable. Larger units of measurement are recommended as they are easy to measure and account for.
Materials and Procedure
The materials needed for this task included water, Pectinase solution, a syringe with a 10cm3 capacity with the plunger removed, a clamp stand, beakers, a stop watch, boiling tubes, a rack for placing the boiling tubes, a water bath set to 30 degrees Celsius, and two measuring cylinders. The Pectinase solution was diluted with water to produce five suitable dilutions. 10cm3 of each of these five solutions or concentrations were made. During the experiment, 6cm3 of pectin in addition to 6cm3 of Pectinase concentration were measured and placed into different boiling tubes. These two boiling tubes were then placed in the water bath for two minutes. The two were then mixed thoroughly by adding the Pectinase into the pectin concentration. The mixture was then incubated in the water bath for about 15 minutes after which 10cm3 of the mixture was poured into a syringe with a thumb over the nozzle. The thumb was removed and the solution drained into the beaker. The time it took for the solution to drain was then recorded. The procedure was repeated for each Pectinase concentration and the results recorded.
Question 1: The use of the 30 degrees Celsius water bath
The temperature of a system affects the activity of an enzyme tremendously. This is because the temperature of a system is usually a measure of a molecule’s kinetic energy. So if the temperature of a system is low, the kinetic energy of that system also decreases. Just as well the temperature of the system increases the kinetic energy of the molecules present in the system. As a result a system has to be of specific temperatures to allow the enzyme for optimum activity. This is because all kinds of enzymes are only active at a specific range of temperature. An increase in temperature results to an increase in the rates of the reaction. There is however limitations to this increase as higher temperatures can lead to the denaturing for the enzyme and therefore, inactivity of the enzyme. The system’s temperature can also not be below the specific temperature for it to be activated. This specific temperature within which the Pectinase enzyme is active is known as the optimum temperature (Jencks 15- 34). The bath therefore, had to be kept at 30 degrees Celsius to allow the Pectinase enzyme to be activated and work at maximum activity.
Question 2: the appropriate control experiment
A controlled experiment is the kind of experiment that uses controls. It usually separates the experiment into two main categories, that is the experimental category and the control category. Ideally, the controlled category is similar to the experimental category, although the experimental entities are changed according to some main variable of interest to the researcher, while the category that is controlled remains unchanged and therefore, constant through out the experiment (Johnson and Besselsen 202- 6). Control experiments have numerous applications and benefits in science. For example, they are very useful in eliminating or reducing any alternate explanations to the results derived from the experiment. For example, an experiment can have several outcomes which can be as a result of several causes. To eliminate each one of these possible explanations or causes to a certain outcome individually can be a daunting task for an experimenter, since it is difficult and time consuming. Instead a researcher can utilize an experimental control in the experiment, which can separate the experimental entities into two separate groups; one that undergoes changes according to the variables of interest and the other group that does not undergo under any changes.
These two experimental groups would however, be kept under identical experimental conditions, and would also be observed in the same manner. With the utilization of a control group, outcomes resulting from a particular change can be attributed to a certain variable with much greater accuracy and confidence (Johnson and Besselsen 202- 6). For this experiment, a control experiment could be performed by having doing the experiment without the enzyme. The pectin in this case would be incubated on its own without being mixed with Pectinase enzyme and then passed from the syringe to the beaker. The measurements of the time the pectin takes to flow through the syringe would also be recorded and compared with those of recorded for pectin and Pectinase mixture. This control group would adequately show the effects of the enzyme on pectin.
Question 3: what is pectin in terms of structure, types of bonds and monomers?
Pectin is a polysaccharide, a carbohydrate that is used by the cell walls of many plant cells as a cementing material. For example, the white rind found in fruits like oranges and lemons contains around 30 percent of this carbohydrate. It is a methylated ester derived from polygalacturonic acid, and it consists around 300 to 1000 chains made of units of galacturonic acid. These units or monomers are joined or linked to each other with 1alpha to 4 linkages. The gelling properties of this carbohydrate are affected by its esterification degree (Buchanan, Gruissem and Jones 26). Below is a part of the structure of pectin;
Question 4: explanation of the graph
Enzymes have three main phases of activity. The first phase is the one where by the catalysis picks pace and the enzymes are the most active. The result is an increasing curve. The second phase is the one whereby the equilibrium has been reached when the rate of reaction has slowed down to a constant rate. The result of this is a plateau like graph. The third phase is when the rate of reaction and the number of active enzymes has slowed down and so the rate of reaction starts to decrease. The result of this is a dropping curve (Jencks 29-46). In the experiment, the higher the concentration of Pectinase, the lower the time the mixture needed to flow down into the beaker. This is because at higher concentrations the rate of reaction is high, the Pectinase enzymes were therefore, very active in clearing the solution and decreasing its viscosity and thus increasing its rate of flow. This is the reason why the curve is dropping with the decrease in concentration of the enzyme.
Question 5: the lock and key model
This model came about to help in explaining the specificity of enzymes. It was suggested that this specificity is as a result of the specific complementary geometric shapes that enzymes and their substrates contain, which fit into each other during a catalytic reaction (Jencks 35-36). This complementarity was then referred to as the lock and key model, because the geometric shapes found in the enzyme and the substrate fit into each other like a key and lock. This is to mean that only a substrate, which is usually the key, of the correct shape and size would fit correctly into the lock, which is the enzyme, and specifically into the active site of the enzyme. The Pectinase enzyme for example, would be the lock containing the active site or the key hole were the key or the pectin molecules would fit in so as to activate the enzyme into action.
Question 6: Genetic engineering/ modification
Genetic engineering or modification is a relatively new trend in science that focuses on manipulating the genome of an organism using modern technology in DNA. This modification involves the introduction into the organism’s genome, foreign DNA or genes that have been synthesized. This has been particularly common in agricultural plants where scientists have modified a number of plants to improve their productivity, enhance their pests fighting activity, and also introduce some other desirable attributes into the crops (British Medical Association 32- 47). Examples of these crops include bananas and tomatoes that have been enhanced genetically to produce fruits that do not ripen rapidly. This is beneficial as it increases the shelf life of the fruits and vegetables, one of the main benefits of this kind of modification. There however, have been concerns about the ability of these genetically engineered crops to cross pollinate with other conventional crops that have not been altered. Other concerns concerning genetically engineered crops have been in regards to the effects such foods might have on the health of consumers. There have been no conclusive research however, that validates claims that genetically engineered foods can be health risks (British Medical Association 32- 47).
Works cited
British Medical Association. The Impact of Genetic Modification on Agriculture, Food and
Health. London: BMJ Books, 1999. Print.
Buchanan, B. B., W. Gruissem and R. L. Jones. Biochemistry and Molecular Biology of Plants.
Rockville, MD USA: American Society of Plant Biologists, 2000. Print.
Jencks, W.P. Catalysis in Chemistry and Enzymology. New York: Dover, 1987. Print.
Johnson, P.D. and D.G. Besselsen. "Practical Aspects of Experimental Design in Animal
Research" ILAR J 43.4 (2002): 202–6. Print.
Novo Laboratories. Enzymes: Nature's catalyst. Teacher's Manual. Wilton, Connecticut: Novo
Laboratories, 1975. Print.
Youde, W. J. Experimentation and Measurement. U.S Department of Commerce, 1997. Print.
