Abstract
In the lab tested, the hypothesis that temperature affects the rate at which substrate is converted into products using the enzyme beta-galactosidase (BGz) and the substrate O-Nitrophenyl-β-D-galactopyranoside (ONPG) The enzyme BGZ is found in a variety of living organisms all the way from bacteria to humans. The byproducts are glucose and galactose. Its common function is to help digest lactose by catalyzing the hydrolysis of the disaccharide into its two components monosaccharides, glucose, and galactose. BGz also can hydrolyze bonds in another B-galactosidase such as ortho-nitrophenyl-galactopyranoside (ONPG). BGz cleaves the galactose off of ONPG resulting in ONP. When the body cannot digest lactose, this results in lactose intolerance. The predicted ideal temperature for the BGz function was about 40°C. The purpose of the experiment is to observe the hydrolysis of ONPG by the enzyme galactosidase catalytic activity of enzymes and determine whether the rate of activity is linear, based on time and temperature. This was done by measuring the effect of temperature on the activity of this enzyme to determine its optimal temperature. The experiment used ONPG and Na2CO3 solution to determine that the optimal temperature for BGz activity in the lab was 40 °C (Table 1; Figure 2). This makes sense because the human body is where these reactions occur, and a human’s body temperature is 37°C. BGz activity was low at cold and high temperatures. Enzyme activity was assessed as a function of temperature using BGz and ONPG as the substrate. The hypothesis was correct, as the rate of ONPG hydrolysis was fastest near 37°C.
Introduction
Enzymes are protein catalysts. Protein catalysts increase the rate of a reaction and by the end of the reaction is regenerated, unchanged, and unconsumed (Bennett-Toomey, Jill). It speeds up protein reactions by lowering the activation energy needed to react with the proteins (Hoefnagels, Mariëlle, and Matthew S. Taylor 78). High concentrations of enzymes increase the speed at which a substrate can be catalyzed (Bennett-Toomey, Jill). The enzymes themselves can be affected by temperature. Higher temperatures increase the kinetic energy of the enzymes but at extreme levels such as 70C, the enzyme can be disrupted, damaged, or denatured (Hoefnagels, Mariëlle, and Matthew S. Taylor 78). Cold temperature lowers kinetic energy of the enzymes and causes little movement or activity compared to optimal temperature.
The galactosidase present specifically in Escherichia coli is a tetrameric enzyme that contains four identical side chains of 1021 amino acids each (Feijoo, S 2). BGz is found in many animals including the human body in the digestive tract specifically. Lactose is a sugar that exists mostly in dairy products. It is a disaccharide, meaning that it is made up of two sugars, galactose, and glucose. In order for organisms to use the sugars, taking advantage of glycolysis, the Krebs cycle, or fermentation, the two units must be hydrolyzed, using water to break them apart (Feijoo, S 2). Beta-galactosidase is the enzyme that specifically hydrolyzes lactose into glucose and galactose. In the case of galactosidase, the enzyme is a protein, made up of amino acids. This enzyme, when immobilized (fixed by physical and chemical forces) on solid supports can be used on a semi‐continuous or continuous basis in bioreactors for milk and other dairy products, and production of sweetener concentrates and food syrups (Feijoo, S 2). The importance of the lab is understanding that without the certain temperature of around 37 degrees Celsius our bodies wouldn’t be able to digest lactose at a reasonable rate. This is important to understand because a majority of human adults are incapable, because of allergy or intolerance) of digesting lactose owing to the deficiency of the enzyme beta‐galactosidase and thus it is a health and nutritional problem. This matters because in 2016 according to the dairy council, “Approximately 70% of the human population has a reduced ability to digest lactose after childhood” (Life cycle 1). This inability to digest lactose is caused by a deficiency of the enzyme b-Galactosidase in the digestive tract, which performs the hydrolysis of lactose to form glucose and galactose, two sugars which are easily absorbed into the bloodstream.
The experiment measured the absorbance of ONPG? at different temperatures to determine its optimal temperature.
For this lab, the hypothesis tested that temperature affects the rate at which the substrate is converted into products using the enzyme BGz and the substrate ONPG. BGz separates the galactose off of ONPG resulting in ONP. ONP is yellow and we can examine how many photons are produced using a spectrophotometer. We predicted that the ideal temperature for the BGz’s function would be about 37C, the human body temperature.
Methods
The BGz enzymes that were in the cell suspension had been derived from E coli. These mixtures were allowed to react for predetermined amounts of time before adding 1.0 mL of 1.0 M Na2CO3 solution to stop the reaction of the enzyme and substrate. Varying temperatures of enzymes were used for the reaction. Water baths were heated up to 0, 4, 35, and 45 °C. The 12 mL cell suspension and ONPG were warmed, while the Na2CO3 stopper solution was left at room temperature at the lab table. There were two duplicate sets labeled “A” and “B” with six samples in each trial. The “1A” and “1B” were blanks. The two blank samples were made to calibrate the spectrophotometer with an absorbance at 420 nm. These samples contained 3.3 mM of ONPG, 1.0 mL of cell suspension, and Na2CO3. Adding Na2CO3 first made sure that no ONPG would be hydrolyzed, denatured immediately, so no reactions could occur, giving us a baseline absorbance reading of solute in all of our reactions (reagent blank). First, two test samples were labeled for each reaction time at the assigned temperature (duplicate). Each experiment had a different temperature assigned. Equal volumes of cell suspension, ONPG and Na2CO3 (sodium carbonate) were used in each reaction. All ingredients were allowed to get to temperature before they were combined for the reactions. The reactions were very time specific so focusing on one trial at a time reduces error. Sodium carbonate (Na2CO3) is a strong base that stops the reaction from proceeding and was added and vortexed to the final solution. This was added at set time points so to determine the rate of enzyme activity at each temperature. The only variable in the experiment was the time of incubation before adding sodium carbonate. Absorbance was measured at 420 nm for each reaction and recorded. Absorbance was converted into moles ONPG hydrolyzed using the following equation: [(absorbance)/ (extinction coefficient)] * volume in liters = mole of product. The extinction coefficient for ONP, the product of the reaction, is 2700 M-1 for the reaction conditions used in the exercise. The extinction coefficient measures the A420 of a 1.0 M solution of ONP. Since Beer’s Law states that absorbance is directly proportional to concentration, the extinction coefficient can be used to calculate -Gz activity in terms of moles of ONPG hydrolyzed. The rate of enzyme activity was compared across the tested temperatures and the optimal temperature was determined 40°C.
Results
The most optimal temperature for BGz reactions to occur over the 20-minute time period was 40°C (Table 1; Figure 2). This is closely related to the human body temperature of 37°C. The human body can withstand being colder more so than being hotter. When the body becomes too hot, components denature quickly. This is proven when the BGz activity remained lowest at 60°C in both trials (Table 1; Figure 1). The least amount of ONPG was present in 60°C because BGz couldn’t thrive, then 0°C was a little more yellow, then 20°C was even more yellow, and 60°C was the most yellow (Figure 1). The yellow coloring is the enzyme?
Discussion
As time went by, the reactions slowed down because it was slowly running out of reactants. Most importantly, as time went by the temperature also cooled so, therefore, the reaction slowed down. The reaction proceeded the fastest at 40°C because that is its optimal temperature. It was almost equivalent to the human bodies 37°C body temperature. At this temperature the enzyme efficiently produces ONPG. The rate of reaction at the coldest temperature very slowly increased. That was because there was minimal kinetic activity occurring so therefore little movement and little ONPG production. The rate of reaction at the hottest temperature was about zero. This was because 60°C was too hot and denatured the enzyme instantaneously. The lab proved that when the enzymes temperature reached over 40°C the enzyme denatures. In this experiment, the hypothesis was found to be correct. Temperature influenced enzyme activity in predictable ways. The higher the temperature the more ONP produced in the reaction, but at very high temperatures enzyme activity was less (less ONP produced). If the reaction was allowed another 20 minutes to go by the reaction at 0°C would slowly increase in enzyme activity and therefore absorbance. This sample would turn more yellow and have a higher absorbance as an extension of the graph (Figure 1). The reaction at 20°C would increase at the same rate and reach the optimal temp of 40°C absorbance in almost double the time (Figure 1). The 40°C sample would continue to absorb at the optimal temperature until the reactants ran out for the reaction. The reaction at 60°C would have little to no activity because the enzyme had already been denatured previously. From the lab analyzed the curve is linear. If the enzyme activity kept reacting eventually the linear curve would plateau because the reaction would consume at the reactants and only have products.
The results of the experiment weren’t surprising. There were no outliers and there was minimal systematic or random error. The random error was human based on measurements. The systematic error could have been caused by not using a Kim-wipe when measuring the absorbance in the spectrophotometer for the blank. Furthermore, the results of the class data matched the overall results from the single experiment.
In the “Journal of Cleaner Production”, the focus was on the BGz activity of commercial lactase samples in raw and pasteurized milk at refrigerated temperatures. The lab relates to the literature cited because of the determination of optimal temperatures for BGz lactose production and BGz pasteurization (Feijoo, S. 2). Both experiment BGz on temperature. Overall Galactosidase has several practical uses. The dairy industry would not be at all what it is without the ability to ferment the sugars from lactose. “Recently, the enzyme has been isolated from bacterial systems to be added into finished dairy products to break down the remaining lactose so that they are lactose-free and do not cause digestive disruption to those suffering from lactose intolerance.” In conclusion, beta-galactosidase is a necessary enzyme to biological systems as a means of utilizing sugars for energy.
If the lab could be redone or extended, it would be curious to study temperatures closer to 40°C. For example, experimenting at the temperatures 35, 37, 39 and 41 °C. From then one would measure the ONPG absorbance to find the most optimal temperature.