The Effect of Increasing Concentration of Enzyme - Lab Report Example

Effect of Temperature and Enzyme Concentration on the Activity of Enzymes The practical was designed to study the effect of increasing concentration of enzyme and change in temperature on the enzyme activity. Rennin (chymosin) was chosen as the enzyme and milk as its substrate since rennin is responsible for the curdling of milk in infants. The experiments very well demonstrated the saturation kinetics of the increasing enzyme concentration. The temperature optima was observed as 37 oC since maximum enzyme activity was observed at this temperature, the decrease as well as increase in the incubation temperature slowed down the enzymatic reaction. At 0 oC as well as 80 oC, no activity was observed. Introduction The two fundamental prerequisites for any form of life are efficient and specialized catalysis of biochemical reactions and the ability to self-replicate. Enzymes are the proteinaceous biological catalysts that carry out biochemical reactions in the biological systems. There are about 2,000 enzymes in our body that participate in different catabolic or anabolic reactions. Enzymes are highly specific in their action and act in different compartments inside the cells and in the extracellular body fluids, e.g., blood clotting factors and the digestive enzymes. Abnormal or absent enzymatic reactions are responsible for a number of diseases or pathological conditions. The catalytic activity of enzymes is not only important for productive intermediary metabolism, recycling and digestion, but is also important for cellular activities such as signal transduction and regulation of cellular functions, e.g., kinases/phosphatases and acetylases/deacetylases; generation of locomotice force, e.g., myosin (heavy chain - a protein serine/threonine kinase; and light chain - calcium/calmodulin-dependent kinase) hydrolyzes ATP to generate force for muscle contraction; and as ion pumps, e.g., ATPases in the cell membrane are involved in active transport. Effect of temperature All enzymes work within a range of temperatures, specific to the medium or organism they work in. The velocity of a chemical or enzyme reaction is almost doubled for every 10o C increase in temperature because the increase in temperature enhances the kinetic energy of the substrates and thus increases proportion of substrates reaching the transition state (Kennel and Rodwell 2006). The rise in enzymatic activity continues till it reaches a maximum, known as the ‘temperature optima’ (Top). The optimum temperature (Top), at which the reaction rate is maximum (Fig.1), generally ranges from 40 to 60o C, but differs from one system to the other, i.e., it is different for human, cold-blooded animals, plants and thermophilic organisms. For most human enzymes, Top is 35-40o C (average 37o C), whereas for most of the plant enzymes, it is 65-70o C. Since life can exist and be sustained at extreme conditions of temperatures, there are examples of enzymes active at very high or extremely low temperatures. Enzymes in the thermophilic organisms may have Top as high as 72o C and are stable up to 100° C (e.g., Taq DNA polymerase). Therefore, Top of an enzyme is mostly the natural temperature of the owner organism. Effect of the enzyme concentration With unlimited amount of substrate and fixed enzyme concentration, the enzyme will be saturated and would operate at the highest velocity (Vmax). Under this condition enzyme concentration becomes rate-limiting factor. Therefore, with increasing amount of the enzyme, the maximum velocity would be a function of the amount of enzyme available. With excess of substrate, and provided that the product is withdrawn, the velocities of the reaction will increase linearly with increasing concentration of the enzyme (Fig. 2). Rennin (optimum pH: 4) Secretion and activation: It is also called chymosin or rennet. Rennin is secreted in large amounts right after the birth and then its production gradually drops off. It is then eclipsed in importance by the Pepsin enzyme. Pepsin and rennin are the only two enzymes produced in the stomach. Rennin is important for infants because it coagulates milk in the stomach to prevent its rapid passage to the intestine. This gives the baby the satiety and sensation of fullness. Rennin is secreted as inactive prorennin that is activated by acid hydrolysis (proteolysis) and by association with calcium ions into active rennin. Actions of rennin: The mechanism is similar to that of pepsin in the presence of calcium. Rennin digests casein (Fig. 3) into paracaseinate, which combines with calcium ions to form insoluble calcium-paracaseinate (milk clot). Latter is then acted upon by pepsin to release smaller peptides (peptones). Milk contains four types of casein. Three of these - α-s1 casein, α-s2 casein and β casein - can be precipitated by the calcium in the milk; calcium causes no reaction on the fourth κ casein. Aim and Objectives Major aim of the experiments was to study the enzyme activity under different conditions of temperature and concentration, i.e. the factors affecting enzyme activity. Specific Objectives To investigate into the curdling potency of rennin enzyme at different temperatures. To investigate the curdling of the milk at different concentrations of enzyme (rennin). Materials and Methods Materials Chymosin (rennin) preparation: The enzyme extracts of genetically engineered microbial culture was provided. Activity – unknown. Milk – Standardised pasteurized milk was provided. 12 test tubes and test tube rack Four water-baths Measuring cylinder / pipette Pasteur pipettes Market pen Timer Methods Preparation: Three water baths were put-on and were allowed to attain different temperatures, viz. 37 oC, 50 oC and 80 oC. Another water bath was set at 0oC with ice. The test tubes were marked from 0 to 11 and 5 ml of milk preparation was added to all. Three test tubes were placed in the water bath set at 37 oC and one test tube each was placed in ice / water baths at 0oC , 50 oC and 80 oC. The remaining 6 tubes in test tube rack were allowed to stand at the laboratory shelf until the milk within them reached room temperature. Experiment 1: Effect of Enzymes concentration on the curdling of milk at room temperature. Experiment was conducted at room temperature in the tubes labelled 0 to 5. Using a Pauster pipette, chymosin preparation (neat, undiluted) was added to the tubes in increasing concentrations, i.e. one drop to tube 1, two drops to tube 2, tree drops to tube 3, four drops to tube 4 and five drops to tube 5. No chymosin was added to the tube labelled ‘O’ (control). The tubes were immediately swirled and timer was started. All the 6 tubes were observed at regular intervals for signs of the milk curdling. The time at which each milk sample started to curdle and the time at which it was completely curdled was recorded. Experiment 2: Effect of Enzymes concentration on the curdling of milk at 37oC. The chymosin enzyme preparation was diluted 1:10 with normal saline. To the three tubes maintained in the 37oC water bath, labelled 6 to 8, chymosin was added – one drop of 1/10 chymosin to tube no. 6, two drops to tube no. 7 and on drop of the undiluted enzyme to the tube no. 8. The tubes were immediately swirled and the timer was started. The time at which each milk sample started to curdle and the time at which it was completely curdled was recorded. Precaution – Addition of the enzyme to the tubes was done quickly so as not to let the milk cool down outside the water bath. All the additions were achieved at fast speed to minimise the time difference between the tubes. Experiment 3: Effect of Temperature on the chymosin activity. To the tubes marked 9, 10 and 11, and maintained at 0oC (tube 9), 50oC (tube 10) and 80oC (tube 11) baths, respectively, one drop of undiluted chymosin was added. The tubes were immediately swirled and the timer was started. The time at which each milk sample started to curdle and the time at which it was completely curdled was recorded. If no curdling was observed in any of the tubes during the time of the lab, a note ‘no curdling observed in tube X after Y minutes’ was recorded in the lab book. Ethical Considerations Microbial rennin (chymosin) is the same enzyme as that purified from rennet, but is extracted from microbes. Thus no calves were harmed during the preparation of this practical. The enzyme preparation has been approved by FDA (1990). RESULTS Experiment 1: Effect of concentration of the enzyme (chymosin) on curdling of milk at room temperature. The results obtained from the three experiments are shown in table 1. Control tube did not show any curdling of the milk till the end the lab time (2 hour). In rest of the tubes (marked 1 to 5) the time taken for curdling appeared to decrease with increasing concentration of the enzyme. Milk in tube no. 1 (one drop of enzyme) took longest to curdle (63.33 min), whereas the tube no. 5 (five drops of enzyme) showed fastest curdling, taking only 28 min for completion of curdling. Table 1: Effect of enzyme concentration on the curdling of milk. Tube No.  0 (control) 1 2 3 4 5 Temp ( oC) RT RT RT RT RT RT Chymosin (No. of drops) 0 1 2 3 4 5 Time taken for curdling of milk 0 63.22 m 42.23 m 33.22 m 29 m 28 m Observations No reaction Limps in middle of the tube (47.34m) and milk curdle completely (solid) at 63.22 m Liquid at middle of the tube (29.49 m) and milk curdle completely (solid)at the side from bottom (42.23 m) Limps in middle of the tubes (27 m) and milk curdle completely (solid) at the side from bottom (33.22m) Milk still liquid in the middle of the tube (20.17 m) and solid at the side of the tube (29 m) Milk still liquid in the middle of the tube (18.21 m) and solid at the side of the tube (28 m) Experiment 2: Effect of concentration of enzyme (chymosin) and temperature on curdling of milk. Results of the experiment are shown in table 2. The enzyme activity at 37oC appeared to be much higher than that observed at room temperature. One drop of the 1/10 diluted enzyme was able to curdle the milk in 29.3 min and two drops only took 7.28 min. Undiluted chymosin preparation showed complete milk curdling in 5.12 min. The enzyme activity appears to be increased almost by 40 times over that observed at room temperature since one drop 1/10 diluted chymosin took almost the same amount of time as taken by 4 drops of neat (undiluted) enzyme at room temperature. Table 2: Effect of enzyme concentration and temperature on the curdling of milk Tube No.  6 7 8 Temp (oC) 37 37 37 Chymosin 1 drop (1/10 diluted) 2 drops (1/10 diluted) 1 drop (Undiluted) Curdling time 29.32 min 7.28 min 5.12 min Observations - At 11.52m the milk start to curdle (soluble) -At the 17.37m milk become more solid but curdling incomplete (less soluble) -At 29.32m the milk curdle completely (solid) -At 2.32m the milk start to curdle (soluble) -At the 4.72m the milk become more solid but not curdled completely (less soluble) -At 7.28m the milk curdle completely (solid) -At 1.52m the milk start to curdle (soluble) -At 3.18m the milk become more solid but not curdled completely (less soluble) -At 5.12m the milk curdle completely (solid) Experiment 3: Effect temperature on curdling of milk by chymosin. The results from the enzyme activity at different temperatures is tabulated in table 3. No curdling of milk was observed in tube no. 9 and 11. The tube no. 10 (50 oC) showed rapid curdling of milk which was completed at 7.64 min. Table 3: Effect of concentration and temperature on the curdling of milk by the action of chymosin ( 0oC, 50oC, 80oC) Tube No.  9 10 11 Temp / oC 00C 50oC 80oC Chymosin (No. of drops) 1 1 1 Curdling Time 0 7.64 min 0 Observation No change Slightly liquid and solid from side to bottom of the tube at 7.64m No change Discussion Results of the above experiments clearly demonstrate the effect of enzyme concentration as well as the incubation temperature on the curdling of milk. Enzyme Concentration - The enzyme activity increased with increasing concentration of the enzyme preparation and hence the time taken for curdling of the milk decreased in proportion with the number of drops of enzyme preparation added to the reaction mixture (Fig. 4). Fig. 4 Effect of enzyme concentration on curdling of milk The figure 1 also demonstrates the saturation kinetics of the enzyme – the enzyme concentration-activity curve is parabolic, i.e. the difference of the curdling time with 4 drops and 5 drops of enzyme is very less compared to that with 1 drop and 2 drops. This kind of enzyme kinetics is known as saturation kinetics, i.e. the active sites of enzyme get saturated after a certain concentration (Fersht A. 1999) Effect of temperature – As shown by the results, temperature of the reaction is highly significant for enzyme activity. The increased activity at increasing temperature is primarily due to increased kinetic energy of the enzyme and the substrate so that there are more number of fruitful collisions between them. In our experiments, the enzyme activity appeared to be increased 40 fold when the temperature was increased form the room temperature (15 oC) to 37 oC. At body temperature, which is 37º C, rate of reaction is most favorable. According to the principles of Kinetic Theory, the increase in temperature imparts the milk and rennin molecules with increased energy and thereby increased velocity. As the molecular movement speeds up, the frequency of molecular collisions goes up and so the rate of chemical reactions increase. Any further increase in temperature beyond the optima would cause a sharp fall in the enzyme activity due to its thermal denaturation and improper substrate binding. Human enzymes start to denature quickly at temperatures above 40° C. Below 37º C, according to the principles of Kinetic Theory, the fall in temperature reduces the energy levels of the milk and rennin molecules. Near or below the freezing temperature, the enzyme is intact but reversibly inhibited because there is not enough heat to overcome the activation energy barrier even for the catalysed reaction. In contrast, chemically catalysed reaction would accelerate with increases in temperature in a linear fashion. But when the temperature is raised above a certain level, it affects and breaks down the enzymes hydrogen and ionic bonds. As the bonds disintegrate, so does the shape of the enzymes active site. The altered active site no longer accepts the caseinogen molecules. The rennin stops affecting them. Sources of Error and Suggestions for improvement The experiment was conducted by taking one tube only for each observation. The use of duplicate (or preferably triplicate) observations could improve the validity of the experiments. Temperature were very extreme, in-between temperatures were missing. More observation points at different temperature could help to give a correct Top. Timing was not very accurate - the experiment may be repeated in order to be more accurate. Dispensing of the enzyme as well as the milk using pipette is not accurate method – Autopippettes could give much more precise results. Water baths can also contribute to errors in the experiment because they maintain the temperature in a given range – repeated calibration of the equipment can improve results. Enzymes distribution within the milk preparation might not be uniform – the enzyme may settle down and make a gradient over a period of time. Incubation on a rotary shaker can improve the enzyme distribution. Conclusions Rennin brings out rapid curdling of milk. The enzyme activity increases with increasing enzyme concentration and the enzyme reaction shows saturation kinetics. The enzyme works very well at 37 oC (the body temperature) and gets denatured at high temperature (80 oC). It does not show any activity at very low (0 oC) temperature. References Fersht A., 1999. Structure and mechanism in Protein Science: A guide to Enzyme Catalysis and Protein Folding. London: Freeman. Kennelly P.J and Rodwell V.W., 2006. Enzyme Kinetics. In: Harper’s Illustrated Biochemistry, Murray R.K., Granner D.K. and Rodwell V.W., Berkshire UK: McGraw-Hill, 27th Ed., 61 – 72. Daniel, R.M., Danson, M.J., Eisenthal, R., Lee C.K., and Peterson, M.E., 2008. The effect of temperature on enzyme activity: new insights and their implications. Extremophiles, 12, 51-59 FDA, 1990. Food Ingredients and Colors. International Food Information Council (IFIC) and U.S. Food and Drug Administration. November 2004; revised April 2010. Available from: http://www.fda.gov/Food/FoodIngredientsPackaging/ucm094211.htm [Accessed 15th Dec.2010]. Read More