Β-Galactosidase Lab Report
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
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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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