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Β-Galactosidase Lab Report

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Introduction: Experiments of the past have confirmed enzyme activity is a significant determinate in the activity of the α-casein with β-casein (Xiaoyu, Yongkang, and Zheng 2012). It’s already been shown before that there are specific enzymes such as Psychromonas ingrahamii that have higher specific activity at lower temperatures. In a separate experiment, it was verified that there was a clear correlation between ortho-Nitrophenyl-β-galactoside (ONPG) concentration and the activity of enzyme beta-galactosidase (β-gal). Higher concentrations of ONPG yielded higher absorbance read by a photospectrometer at 421nm. Since Exercise 1 was conducted under standard atmospheric conditions of a standard classroom setting, further experimentation was profitable to determine whether or not temperature had a significant impact on enzyme activity or not. In order to find the answer, an experiment was conducted in which 7 test tubes filled with equal amounts substrate and enzyme were subjected to various temperatures (4˚C, 22˚C, 37˚C, 45˚C, 65˚C, and 95˚C) for ten minutes. It was hypothesized by the group that difference in temperature will have a significant effect upon enzyme activity based on past knowledge on the anatomy and …show more content…

For the remaining 6 tubes, 2mL Buffer A and 500 µL 5 mM ONPG was added and mixed, keeping the ratio of Buffer A to ONPG constant. Immediately following the preparation of each tube, as an independent variable, they were all placed into temperature baths of 4˚C, 22˚C, 37˚C, 45˚C, 65˚C, and 95˚C for a total of ten minutes. The 22˚C tube was left to incubate at room temperature. After incubation, 750µL of Buffer B was added and mixed in, in order to halt the reaction in each tube. The absorbance level of each was read with a photospectrometer at 421nm, with the blank used as a zero. These absorbance values were used as the dependent

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The substrate for beta-galalactoside is ortho-nitrophenyl-B-galactoside. ONPG is structured similarly to lactose. The purpose of the experiment was to add a competitive inhibitor to observe if the reaction rate would slow down. A competitive inhibitor is when the inhibitor binds to the active site on the enzyme and prevents the binds of the substrate

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Use these results to determine the product concentration, using Beer-Lambert’s Law: A= ɛCl (where A is the absorbance, ɛ is the molar absorptivity, C is the product concentration and l is the length of solution that the light passes through). Calculate the product concentrations at every minute for 10 minutes for all 7 of the test tubes using Beer-Lambert’s Law. Plot a graph of product concentration vs. time and then use the gradients of the 7 test tubes to determine the velocities of the reaction. After calculating the velocities, plot a Michaelis-Menten graph of velocity vs. substrate concentration.

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The absorbance level @ 520 nm obtained from the spectrometer indicates the amount of urea obtained via measuring the absorbance of the light through the supernatant coloration, which was provided by the

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