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Year:: 2019
Contributor:: Patel, Rishi (Creator); Li, Silei (Creator); Lorence, Nicholas (Creator); Veintimilla, Alison (Creator)
Subject:: Glutamate; Aspartate; Escherichia coli; β-galactosidase; Beta-galactosidase; Enzymes
DOI: https://doi.org/10.26300/vxp0-mb40

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Abstract:: Escherichia coli β-galactosidase is an important homolog of the human β-Galactosidase and can be used to understand the function and mechanism of its breakdown of lactose to glucose and galactose. To understand the function of the magnesium metal binding site 419 on the function of the enzyme, we conducted a site-directed mutagenesis H419P. However, due to an unintended insertion of the mutagenesis primers in the DNA, we were unable to continue this experiment. However, we obtained pre-transformed E537D E. coli β-galactosidase expressing BL21STAR cells. Glutamate 537 is an essential residue present in the active site of E. coli β-galactosidase because it acts like the nucleophile to bind the substrate. Glutamate and Aspartate only differ by one carbon hence this mutation can be used to understand the significance of Glu-537 for the function of E. coli β-galactosidase. We measured the rate of reaction of the E537D mutant E. coli β-galactosidase on ONPG and compared it to that of the wild-type E. coli β-galactosidase. We predicted that the mutant E537D would have decreased catalytic activity in comparison to the wild-type. Based on the results of the study, we created Michaelis-Menton graphs and Lineweaver-Burk plots to calculate the Vmax, Km, kcat and catalytic efficiency, all of which decreased from wildtype to the mutant. We saw that there was an approximately 600-fold decrease in the kcat of the mutant, indicating a drastic reduction in the rate of the reaction for the mutant E537D. Hence, we concluded that Glutamate is a very important residue for the catalytic activity of E. coli β-galactosidase.

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Use and Reproduction: This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License

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Patel, Rishi, Li, Silei, Lorence, Nicholas, et al., "Impact of Glutamate to Aspartate Substitution on Catalytic Activity of β-galactosidase (Escherichia coli)" (2019). Undergraduate Course-Based Research Poster Fair Collection. Brown Digital Repository. Brown University Library. https://doi.org/10.26300/vxp0-mb40

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Undergraduate Course-Based Research Poster Fair Collection

In an initiative supported by HHMI and the Sheridan Center, Brown faculty developed or modified courses under the Course-based Undergraduate Research Experiences (CURE) model. In a CURE course, students pursue a novel research project in the classroom setting. This collection …
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Impact of Glutamate to Aspartate Substitution on Catalytic Activity of β-galactosidase (Escherichia coli)