Lactase Enzyme Activity - Lab Report Example

Lactase Enzyme Activity Introduction Enzymes are biological molecules made up of a complex protein that catalyze specific chemical change in living organisms. In this regard, enzymes are capable of regulating the rate at which chemical reactions occur without being altered in the process. Enzymes achieve this function by lowering the activation energy necessary for reaction to occur in cells. The minimum energy that is required for a chemical reaction to occur and yield products is called the activation energy. Even though reaction can still occur within the cells and yield products, biochemical reactions without enzymes often take place at a very much lower rate. Reactants often referred to as substrates are bound with enzymes through the enzyme’s active sites. According to Zheng et al (2012), active sites within an enzymes are flexible thus after binding to a substrate and products formed, the active site is unbound and return to its original shape. Lactase enzyme is an enzyme found within the intestine, kidney and liver and mammals. Lactase catalyzes the breakdown of lactose into its sub units namely D-glucose and D-galactose that can be utilized by the body as a source of energy within the cells (Steinhoff-Wagner et al, 2014). In this regard, individuals who lack lactase enzyme are not capable of metabolizing lactose and are referred to as lactose intolerance hence are supplied with enzymatic supplements. Enzyme activity is a very important process in cell of living organisms. This is because it is within the cell where all biochemical reactions takes place and any alterations to its activity will definitely alter the functions of that particular cell. As stated by Jing et al (2009), enzymatic activity can be affected by altering the environment within which a reaction is taking place. In this study, we examined how the enzymatic activity is affected by temperature, pH, Co-factors and enzyme specificity. Through the use of lactase solution, this experiment aims at testing chemical and physiological properties of lactase enzyme. Based on my knowledge of the effects of temperature and pH on the enzymatic activity of lactase, if the lactase used in today’s lab was extracted from human cells, I hypothesize that lactase enzyme activity will be optimal at temperature and pH of 400C and 8 respectively. Materials and Methods Effects of temperature on Lactase enzymatic activity Microfuge tubes were selected and labelled according to the selected temperatures 00C, 250C, 400C, 600C, 800C and 1000C and each filled up to 0.5 lines with lactase solution. The solutions were then maintained at water baths with respective temperatures for five minutes after which milk was added to lactose solution up to 1.0 line using alternate plastic pipette. The solution was left for ten minutes after which a glucose strip was placed into each of the test tubes for one second and allowed to sit on the bench for thirty seconds. The coloration of the glucose strip was then compared with the chart and the amount of glucose recorded in mg/dL. Effects of pH on Lactase Enzyme activity Seven microfuges were selected and labelled; 2, 4, 5, 6, 7, 8, 10, and 12, filled with appropriate pH buffer up to 0.5 line and 3 drops of milk added into each tube. The solution solutions in the seven tubes were then mixed by inverting the tubes three times after which 3 drops of lactase solution was added to each tube using a plastic pipette. All tubes were then incubated for 10 minutes in a water bath at 400C. A glucose strip was then placed in each test tube for one second, removed and left on the bench for thirty seconds and finally coloration compared to the chart provided. Lactase Enzyme Specificity Two microfuge tubes were selected and labelled “L” and the other labelled “M”. Lactose and Maltose solutions were thenadded into the tubes labelled L and M respectively up to the 0.5 line. Lactase solution was then added into each tube up to 1.0 line and the mixture placed in 400C water bath for 10 minutes. After ten minutes, a glucose strip was then placed into each tube for one second, removed and placed on the bench for 30 seconds and coloration finally compared to the chart provided and amount of glucose determined recorded in mg/dL. Effects of Inhibitors on Lactase Enzymatic Activities Two microfuge were selected and labelled control and EDTA respectively. 0.5M EDTA was then added to the tube labelled EDTA up to the 1.5 line while on the control, distilled water was added until it reaches the 1.5 line on the tube. Three drops of milk were then added to each tube, inverted to mix and allowed to sit for one minute. 3 drops of lactase solution were then added into each tube and both tubes placed in a water bath at 400C for 10 minutes. A glucose strip was then placed on each tube for one second, removed and allowed to sit on bench for thirty seconds then coloration compared to the chart provided to determine the amount of glucose in mg/dL. Results Table 1: Effect of Temperature on Enzymatic Activity Temperature (0C) O 25 40 60 80 100 Glucose mg/mL 0 600 1000 800 300 0 Figure 1: Effects of temperature on Lactase enzyme activity Table 2: Effects of pH on Lactase Enzyme Activity pH 2 4 6 7 8 10 12 Glucose (mg/mL) 0 50 400 750 1000 50 0 Figure 2: Effects of pH Lactase Enzyme Activity Table 3: Lactase Enzyme Specificity Lactose Maltose Glucose (mg/mL) 950 50 Table 4: Effects of Inhibitors on Lactase Activity Control EDTA Glucose (mg/mL) 950 50 Discussion The aim of this experiment was to determine how enzymatic activity is affected by temperature, pH, Inhibitors and specificity of the enzyme. In this study enzyme is lactase solution derived from animal while the substrate was lactose derived from milk samples. With regard to this experiment, the independent variable is the temperature while dependent variable is concentration of glucose in mg/mL. This is because lactase enzyme breaks down lactose into its sub units namely D-glucose and D-galactose which are the products of this enzymatic activity (Zheng et al, 2012). Temperatures below 400C show low enzymatic activity with activity increasing up to maximum of 1000mg/dL of glucose at 400C. However, further increase in temperature results into loss of lactase enzyme activity with 0mg/dL at 1000C as shown in table 1 and figure 2 respectively. The loss in enzyme activity at temperatures above 400C is because enzymes are denatured at these temperatures hence active sites are lost (Steinhoff-Wagner et al, 2014). pH affects activity of lactase enzyme just like the temperature. However, lactase enzyme is more specific with regard to pH changes. At pH above and below 8 there was drastic reduction in enzyme activity as shown in table 2. The loss of enzyme activity is because of the fact that lactase enzyme is denatured at pH above and below 8 as shown in figure 2. As stated by Prykhodko et al (2015), lactase enzyme is protein in nature and one of their characteristics is that they have active sites that bound with substrate thus when they are denatured they remain dysfunctional because they lack ability to bind with the substrate. As stated by Steinhoff-Wagner et al (2014), lactase enzyme is found within the small intestine, kidney and liver where pH tends to be slightly basic. Enzymes are very specific to their substrate and can only catalyze certain biological reactions and not others. As stated byIllanes (2008), enzymes being globular in shape and have at least one active site each normally structured so that it binds with specific substrate as in a lock and key structure. EDTA is an inhibitor and that is why the use of EDTA results into loss of enzyme activity as shown by reduction in glucose production as shown in table 4. As stated by Bettelheim(2007), cofactors are inorganic compounds often metallic ions that are part of active sites of enzymes. The formation of substrate enzyme complex is made easier with the availability of lactase enzyme cofactors such as magnesium and copper. However, EDTA as an inhibitor binds magnesium and copper thus makes them unavailable for utilization as cofactor. Conclusions After comparing the effects of temperature, pH and the presence of inhibitors on the production of glucose, I conclude that 400C and pH 8 are the optimal temperature and pH respectively where lactase enzyme works best. Consequently, lactase enzyme is specific to lactose hence lactose is the best substrate for this enzyme compared to maltose. It is also evident that inhibitors such as EDTA results into loss of lactase enzyme activity. The results of this study also confirms my hypothesis that lactase enzyme is derived from animal. References Bettelheim, F. A., March, J., & Brown, W. H. (2007). Introduction to organic and biochemistry. Belmont, CA: Brooks/Cole Illanes, A. (2008). Enzyme Biocatalysis: Principles and Applications. New York: Springer Jing, M. Y., Sun, J. Y., Weng, X. Y., & Wang, J. F. (2009). Effects of zinc levels on activities of gastrointestinal enzymes in growing rats. Journal Of Animal Physiology & Animal Nutrition, 93(5), 606-612. Prykhodko, O., Pierzynowski, S. G., Nikpey, E., Arevalo Sureda, E., Fedkiv, O., & Weström, B. R. (2015). Pancreatic and Pancreatic-Like Microbial Proteases Accelerate Gut Maturation in Neonatal Rats. Plos ONE, 10(2), 1-14. Steinhoff-Wagner, J., Zitnan, R., Schönhusen, U., Pfannkuche, H., Hudakova, M., Metges, C. C., & Hammon, H. M. (2014). Diet effects on glucose absorption in the small intestine of neonatal calves: Importance of intestinal mucosal growth, lactase activity, and glucose transporters. Journal Of Dairy Science, 97(10), 6358-6369. Zheng, Y., Liu, J., Ma, Y., Xu, Y., Xu, F., & Hua, T. (2012). Temperature Effects on Enzyme Activity of Chicken Liver Esterase Used in Calorimetric Biosensor. Artificial Cells, Blood Substitutes & Biotechnology, 40(1/2), 125-13 Read More