Latase Lab Report

Lactase is an enzyme that splits up the disaccharide lactose into monosaccharides glucose and galactose, but its function diminishes with age resulting in a wide use of lactase supplements. This enzyme functions under specific conditions, so we investigated the effect different salt concentrations have on the enzyme activity. Serial dilutions were performed to prepare a lactase solution as well as twenty percent, fifteen percent, and five percent concentrations of NaCl. Three different treatments of ortho-nitro-phenyl-galactoside, lactase, and perspective salt concentrations were prepared in cuvettes where the reaction would be observed and recorded from a spectrophotometer. Larger concentrations of salt adversely effect the reaction activity
of lactase, slowing it down. When comparing concentrations to a control, a middle moderate amount of salt (15%) yields a reaction rate close to that of a normal reaction absorption (0.799) without the presence of salt. Smaller concentrations (5% concentration) or larger concentrations (20% concentration) salt however, is what slows down the reaction rate.
It is concluded that those whose lactase functions have diminished should monitor their salt intake. Depending on the severity of the intolerance, salt may have to be eliminated from the diet; however, in moderation lactase supplements and the lactase enzyme can function in the presence of salt, but not as efficiently.
Introduction
Enzymes are produced by living organisms as catalyst in which accelerate chemical reactions. Enzymes act upon substrates, transferring them into differing products. Enzymes function under particular conditions for optimal functionality. Lactase is an enzyme produced by cells that line the wall of the small intestines. This enzyme helps to digest lactose, a sugar found in dairy products, and break it into glucose and galactose. Glucose and galactose are smaller sugars that are easier to absorb. As infants, lactase activity is the highest and best functioning due to milk being the main source of nutrition. As one ages, lactase activity decreases causing older peoples bodies to be unable of tolerating large amounts of lactose (Rings et al., 1994). This is referred to as primary lactase deficiency and is the most frequent form of lactose
intolerance. Genotypes and ethnic backgrounds have a considerable effect on decrease in lactase activity with age (Strzałkowska, Jasinska, & Jazwik, 2018). Treatments for lactose intolerance include lactose treated dairy products such as lactaid, lactase supplements, limitation of lactose, and/or elimination of lactose containing food from the diet (Heyman, 2006). Due to unique amino acid chains, enzymes are made for specific purposes to catalyze under specific conditions. One thing in which can influence the activity of enzymes, such as lactase, is salt concentration. Salt is an ionic compound whose molecules disassociate in water. They have individual and opposite charges. These charges can have an affect on the charges of the amino acid chains that enzymes are built of. If salt concentrations effect enzyme activity, then different concentrations will disrupt the normal reaction rate of lactase catalyzing lactose. Lactase supplements, called lactaid, are treatment options for those who are lactose intolerant; however, if a supplement is taken prior to a salt and dairy heavy meal how effective will the supplement be. The body has a natural salt concentration; however, the body is not always in a homeostatic state. Therefore, when lactose is ingested lactase must catalyze it while NaCl ions disassociate using more energy. The results can show an appropriate salt concentration intake for those who have lower levels of lactase activity.
Materials and Methods To begin, a lactaid pill was crushed into a powder using a mortar and pestle. The powder was then transferred to a beaker containing 10 ml of 0.1 M phosphate buffer where it was left to dissolve. After 1-2 minutes the solution was filtered into a clean small beaker using a paper towel so that any pieces of the pill that remained would not be in the enzyme stock solution. Next a serial dilution was done to get a 1/100 lactase concentration. This was done by adding 500 micro milliliters of the stock enzyme to a test tube containing 4.5 ml of PO4 buffer, resulting in a 1/10 concentration. 500 micro milliliters of the 1/10 concentration was then added to another test tube containing 4.5 ml of PO4 buffer. From here 20%, 10%, and 5% NaCl concentrations were determined to be used as the independent variables in each trail. To achieve these percentages another serial dilution from the concentrated 20% NaCl had to be made. To do this 2 ml of 20% NaCl was taken and put into a test tube containing 2 ml of DI water resulting in the 10 percent concentration. Next 2 ml was taken from that test tube and added to another test tube containing 2 ml of DI water resulting in the 5 percent concentration. Lastly, another 2 ml was taken from the 5 percent concentration, but this 2 ml was trashed not used. After making the concentrations the cuvettes needed to be prepared to run 3 different trails using the 3 different concentrations of salt. Before starting that, a control had to be tested. A cuvette was prepared using 1 ml of the 1/100 concentration enzyme stock, 1 ml of 2.5mM Ortho-nitro-phenyl-galactoside, and 1 ml of PO4 buffer. The enzyme activity is measure via the release of the yellow color released during the reaction and production of D-galactose and O-nitrophenol. Three milliliters of PO4 buffer was put into an empty cuvette to serve as the blank for the Genesys 20 spectrophotometer. After blanking, the control was put into the spectrophotometer at 420nm and the light absorption was calculated every 15 seconds for 3 and half minutes. This was done twice so that the average and difference in absorption could be calculated. All numbers were recorded in a table. To prepare the cuvettes with the three different concentrations of salt, 1 ml of 1/100 concentration of stock enzyme, 1 ml of 2.5mM ONPG, and 1 ml of respective NaCl concentration (20,10, and 5) was added to individual cuvettes similar to the control. Each concentration was tested twice in the spectrophotometer resulting in six trails. The average absorption and difference in absorption was calculated from the table and put into a graph.
Results
As stated before, it was expected that increased concentrations of NaCl would negatively effect enzyme activity. As seen in figure one, when the sample with the greatest concentration was tested the absorption was at its lowest with an average of 0.500. While the 10 percent and 5 percent solutions were around this number, all were much lower than the average absorption of the control which was 0.799. This shows a significant decrease in absorbance with any amount of NaCl present. The average change in absorbance, shown in figure 2, demonstrates the decrease in the reaction rate by the decrease in light absorbed. All three solutions yielded similar results with their averages all being close together.
Discussion
The reaction activity of lactase decreased in the presence of different concentrations of salt.
The twenty percent concentration of salt yielded the largest decrease in enzyme activity. On the other hand, the fifteen percent concentration had the closest reaction time to the control, but still demonstrated a significant decrease. Due to these results the hypothesis was not supported. It was expected that the smallest concentration of salt, five in this experiment, would yield reaction rates similar to those of enzyme activity without the presence of the salt. This may be a result of the density of the salt ions. The discrepancies between the fifteen percent being closer to the control compared to the five percent maybe the results of an optimal salt level being reached (Lanyi & Stevenson, 1969). Salt concentrations seem to negatively effect enzyme activity in extreme numbers. In moderation, salt concentrations can stimulate enzyme activity (Lanyi & Stevenson, 1969). NaCl does affect enzyme activity, but not always negatively. Furthermore, those who are lactose intolerant should monitor their salt intake. Moderate salt intakes are different for each person, so there is no exact recommended amount. If one is consuming lactase supplements to aid in digestion, it is best to avoid adding salt at the same time because it may alter the job of the lactase supplement thus altering the effectiveness. Future studies should include different types of salt with differing
concentrations. These can include sea salt, iodized salt, non-iodized salt, and kosher salt. The amount of lactase also could be quantified more specifically rather than just a concentration amount. These studies would help those who are lactose intolerant find a better option for salts when using a lactase supplement to aid in digestion.