Effects of Temperature and Enzyme Concentration on Enzyme Activity - Lab Report Example

Abstract Enzyme concentration and temperature affect the rate of enzyme catalyzed reactions. The investigation of the effect of temperature and enzyme concentration can be done by varying various variables such as the concentration of the enzymes, and temperature. Chymosin can be used in the investigation of the rate at which milk coagulates at specific temperatures and over a range of temperature. The information derived from the time taken for the milk to completely coagulate is necessary in determining the effect of enzyme concentration and temperature on enzyme activity. Increase in enzyme concentration leads to a rise in the rate of enzyme catalyzed reaction. On the other hand, increase in temperature leads to increased rate of reaction. However, temperatures above the optimum for a given enzyme denature them hence reducing the reaction rate. Introduction Enzymes are biological catalysts that enhance chemical reactions (Whitehurst and Oort 2009). Just like other catalysts, enzymes work by lowering the activation energy for a reaction. According to Pratiyogita Darpan (2005), enzymes reversibly combines with substrates through their active sites forming enzyme-substrate complexes that eventually break down into products after the enzymes have dissociated from the substrates. This can be expressed as a single equation using symbols as shown below: E + S ES E + P (where E = enzyme, S= substrate, and P= products. The symbol = indicates that the reaction is reversible). The velocity of enzyme catalyzed reaction, however, varies with changes in temperature, enzyme concentration, substrate concentration, and pH. These four factors affect enzyme activity, which Dee and Stoker (2009) define as the measure of the rate at which enzymes convert substrate to products in a biochemical reaction. This report, however, discusses an investigation only on the effects of temperature and enzyme concentration on enzyme activity. In this experiment, we investigated the effect of concentration of chymosin (an enzyme) on the rate at which milk coagulated at specific temperatures (i.e. room temperature and 370 C). We also investigated the effect of varying temperature on the rate of milk curdling in the presence of chymosin enzyme. Chymosin is a microbial rennin same as those purified from rennet although the former is extracted from microbes. The report presents the experiment procedures, the results obtained and an analysis as well as discussion of those results. Methods Twelve milk portions were prepared by measuring 5 mL of milk into 12 test tubes. 3 of the test tubes were placed in a 37°C water bath, 1 in a the 0°C ice bath, 1 in a 50°C water bath and another in a 80°C water bath. The remaining 6 test tubes were left in the test tube rack until the milk contained in them reached room temperature. The investigation on the effect of enzyme concentration on enzyme activity was done on two folds (first at room temperature and the other at 37°C). Investigation for the effect of enzyme reaction at room temperature involved the use of six test samples in the 6 test tubes that were left at room temperature. These test tubes were numbered from 0 to 5. One drop of neat chymosin was quickly added to tube labeled number 1 using a Pasteur pipette, two drops to tube number 2, three drops to tube number 3, four drops to tube number 4, and five drops to tube number 5. Tube labeled number 0 was used as a control and no chymosin was added to it. The six test tubes were swirled immediately and the timer started. The tubes were observed at regular intervals for signs of milk curdling and the time taken for each of the milk sample to completely curdle was recorded. For the investigation of the effect of enzyme concentration on rate of reaction at 37°C, the 3 samples in the test tubes placed in a 37°C water bath were used. They were numbered from 6, 7 and 8. One drop of 1/10 diluted chymosin was quickly added to tube labeled 6 using a Pasteur pipette, two drops of diluted chymosin to tube number 7 and lastly, one drop of neat chymosin to tube number 8. The three test tubes were swirled immediately and the timer started. The time of removing the tubes from the water bath was minimized and the swirling was done quickly to avoid the cooling down of the milk. Similarly, the tubes were observed at regular intervals for signs of milk curdling and the time taken for each of the milk sample to completely curdle was noted. Finally, the experiment to determine the effect of temperature on enzyme activity involved adding one drop of neat chymosin to the three remaining tubes (in the 0°C water bath-tube 9, 50°C water bath-tube 10, and 80°C water bath- tube 11). These test tubes were swirled immediately and the timer started. The time of removing the tubes from the water baths was also minimized and the working was done quickly to avoid the cooling down of the milk. Observations were made and the time taken for cuddling to occur was recorded. Results Table 1 shows the class results for the effect of enzyme concentration and temperature on the curdling of milk in the presence of chymosin. Table 1. Class results for the experiments. No. drops of chymosin 0 1 2 3 4 5 1 drop 1/10 2 drops 1/10 1 1 1 1 Temperature Room Room Room Room Room Room 37 C 37 C 37C 0 C 50 C 80 C   Time taken for 5 mL of milk to coagulate / seconds   Group 1 Nothing Nothing 2160 2091 1982 1740 863 293 153 Nothing 41 Nothing Group 2 Nothing Nothing 2165 2100 1958 1826 1126 779 300 Nothing 42 Nothing Group 3 Nothing 3320 2910 2460 2430 2400 930 460 375 Nothing 50 Nothing Group 4 Nothing Nothing Nothing Nothing Nothing Nothing 834 390 60 Nothing 100 Nothing Group 5 Nothing Nothing 3420 3780 3630 3512 1440 2100 240 Nothing 70 Nothing Group 6 Nothing 3529 3335 2435 2145 1745 930 565 250 Nothing 90 Nothing Group 7 Nothing Nothing Nothing 3420 3410 3050 635 440 440 Nothing 120 Nothing Group 8 Nothing 3180 2760 2364 2051 1925 1842 632 550 Nothing 54 Nothing Group 9 Nothing Nothing 3520 3128 3024 2559 772 581 171 Nothing 16 Nothing Group 10 Nothing Nothing Nothing 2400 2200 2040 600 780 120 Nothing 85 Nothing Group 11 Nothing 3287 3420 2990 2700 2680 530 1935 530 Nothing 28 Nothing Group 12 Nothing Nothing 4320 3960 3180 2940 360 720 130 Nothing 55 Nothing Group 13 Nothing 3500 3350 2940 2520 2420 1250 865 456 Nothing 72 Nothing Average Nothing 3363 3136 2839 2603 2403 932 811 290 Nothing 63 Nothing Table 2 below shows my results for the determination of the effects of enzyme concentration and temperature on the curdling of milk in the presence of chymosin. Table 2. My results. Test Tube 0 1 2 3 4 5 6 7 8 9 10 11 No. drops of chymosin 0 1 2 3 4 5 1 drop 1/10 2 drops 1/10 1 1 1 1 Temperature Room Room Room Room Room Room 37 C 37 C 37 C 0 C 50 C 80 C  Results Time taken for 5 mL of milk to coagulate   Minutes X52 X52 X52 43.39 45.60 46.01 58.34 9.47 9.47 X59.10 2.4 X59.10 Seconds X3120 X3120 X3120 2603.4 2736 2760.6 3500.4 568.2 568.2 X3546 144 X3546 The variables involved in determining the effect of enzyme concentration on rate of reaction are enzyme concentration (independent), time (dependent), temperature was held constant in the two experiments (room temperature and 370C) ; and in experiment to determine the effect of temperature on enzyme activity include time (dependent) and temperature (independent), enzyme concentration remained uniform. Table 3 below shows the range, standard deviation and variance of the class results and my individual results. Table 3: ranges, standard deviation and variance for the above data Group Range (Time in seconds) Standard deviation Variance Group 1 2119 924.6406155 854960.2679 Group 2 2123 843.5295 711542 Group 3 3270 1239.833 1537186 Group 4 774 357.021 127464 Group 5 3710 1543.342 2381905 Group 6 3439 1290.639 1665750 Group 7 3300 1554.008 2414942 Group 8 3126 1067.764 1140119 Group 9 3504 1469.977 2160833 Group 10 2315 1007.245 1014542 Group 11 3392 1314.295 1727371 Group 12 4265 1816.35 3299128 Group 13 3428 1289.916 1663883 SELF 3356.4 1360.232 1850232 Graph 1: Effect of enzyme concentration on rate of reaction at room temperature for class and individual results. Graph2: Effect of enzyme concentration on rate of reaction at 37°C for class and individual results. Graph 3: Effect of temperature on enzyme activity –a plot of the reciprocal of time against temperature. Discussion Considering the relationship between the rate of reaction and the concentration of chymosin enzyme in the investigation of the effect of enzyme concentration on rate of reaction at room temperature, it was noted that initially they are directly proportional to one another. An increase in enzyme concentration leads to a rise in the rate of chemical reaction. Were tubes 1 and 2 left for some extra time, coagulation would have taken place hence follow the same trend just like the class results from various groups. This can be justified from the results obtained when working with milk samples at 370 C (refer to graph 2). The same trend is depicted i.e. a rise in reaction rate with rise in enzyme concentration. Under normal circumstances, the concentration of enzymes is low as compared to the concentration of the substrate (Seager, and Slabaugh 2010; Warren, Goodman and Attridge2006). Therefore, an increase in the enzyme concentration increases the ES in compliance with the reaction rate theory: E + S =ES (i.e. increased E gives more ES). This is explained by the availability of more enzyme molecules that catalyse the reaction giving more ES and thus a higher reaction rate. According to (Pratiyogita Darpan 2005), doubling of the enzyme concentration doubles the rate of conversion of substrate to products. However, in our case we used a limited sample. This means a further increase in the enzyme concentration after the initial stage will have no effect to the rise of reaction rate, but instead will reach a maximum where it will the level off. A comparison between the reactivity rates with increase in concentration for both room temperature and 370C show that reactivity is higher with a rise in temperature. Similarly, from graph 3, there is a rise in rate of reaction as the temperature rises from room temperature to 500 C through 370C. There were no observable changes to the milk samples at 00 C and 800 C for both class groups and my results. This is in line with other investigations in the area that indicate how velocity of enzyme catalysed reactions increase with temperature rises, however, over a limited range of temperatures (Newsholm and Leech 2010; Clark 2007; Malone and Dolter 2008). According to Wolberg, Hong, Dougald and Maureane (2004) and Hames and Hooper (2000), there is an optimal temperature at which an enzyme catalyzed reaction is at its maximum. For our case we can use 500 C as the optimum temperature. Although, the experiment was not carried out at several minimum temperatures to indicate an accurate optimal temperature, we assume that beyond 500 C, the reaction dropped sharply. This is attributed to the denaturation of chymosin (protein enzymes) by heat and that why there was no observable change in the milk sample at 800C water bath. Chatterjea (2012) argues that enzyme activity progressively decreases when temperatures are below the optimum temperature for the specific enzyme and that why there were no observable changes to tube 9, even after waiting for 3546 seconds. At 0 0C, the enzymes become inactive and this behavior was similar to the experiments by various class groups. To improve the outcomes of similar experiments, it could be advisable to consider using several temperature points to depict the exact trend in the effect of temperature on enzyme activity. More time should also be allowed for samples placed at lower temperatures so as to give the accurate data for the investigation. Conclusion Enzyme concentration and temperature are some of the key factors that affect enzyme activity and consequently the rate of enzyme catalysed reactions. Increase in enzyme concentration leads to a rise in the rate of enzyme catalyzed reaction. A further increase in the concentration results into the rate of reaction reaching its maximum where it finally levels off. Considering temperature, it is noted that an increase in this variable leads to an increase in the enzyme catalyzed reactions, however for a limited temperature range until it reaches the optimum. Any temperatures past this optimal leads to denaturation of the enzymes, and thus a sharp reduction in the reaction rate is noted. List of References Chatterjea, 2011. Textbook of Medical Biochemistry. Australia, Melbourne: JP Medical Ltd. Clark, J. 2007. The Effect of Changing Conditions in Enzyme Catalysis [online]. Available at http://www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html [Accessed 20 February 2012]. Cornish-Bowden, 2004. A Fundamentals of enzyme kinematics, 3rd ed. New Jersey, NJ: Portland Press. Dee, J and Stoker, H. S 2009. Organic and Biological Chemistry. Mason, OH: Cengage Learning. Hames, N. Hooper, M 2000. Instant Notes in Biochemistry. London: Garland Science. Malone,LJ and Dolter, T 2008. Basic Concepts of Chemistry , New York, NY: John Wiley and Sons. Newsholme, E and Leech, A 2010. Functional Biochemistry in Health and Disease. New York, NY: John Wiley and Sons. Nord, F and Werkman, CH 2006. Effects of Temperature on Enzyme Kinetics. In Eric J. Toone (ed). Advances in Enzymology and Related Areas of Molecular Biology, Volume 3. Sydney: John Wiley and Sons. Pratiyogita Darpan 2005. “Enzyme Action and Inhibition,” Competition Science Vision, 8(92), pp. 1091- 1097. Seager, S L and Slabaugh, M, R. 2010. Chemistry for Today: General, Organic, and Biochemistry. Mason, OH: Cengage Learning. Warren, D., Goodman, N and Attridge, E. 2006. The Essentials of GCSE OCR Additional Science for Specification: A. Text. London: Letts and Lonsdale. Whitehurst, RJ and Oort MV, 2009. Enzymes in Food Technology. New York, NY: John Wiley and Sons. Wolberg, AS., Hong MZ., Dougald, M and Maureane, H 2004. “A Systematic Evaluation of the Effect of Temperature on Coagulation Enzyme Activity and Platelet Function,” Journal of Trauma-Injury Infection & Critical Care, 56(6), pp. 1221-1228. Read More