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Year:: 2021
Contributor:: Glik, Masha (Author); Gallagher, James (Author); Chin, Logan (Author)
Subject:: Escherichia coli; β-galactosidase; Beta-Galactosidase; Enzymes; E. coli; G794N
DOI: https://doi.org/10.26300/58qg-k302

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Abstract:: While lactose intolerance is globally on the rise, beta-galactosidase enzyme has gathered industrial attention due to its lactose digesting properties. E. coli β-galactosidase is an important homolog of the human β-galactosidase and can be used to unravel the mechanism behind the breakdown of lactose to glucose and galactose. To understand the role of the 794 residue in the active site loop in the function of the enzyme, we conducted a site-directed mutagenesis G794N to differentiate between the steric and electronic effects of the 794 residue on the enzyme function. Mutant G794N E.coli β-galactosidase protein was expressed in BL21STAR cells. We measured the rate of reaction of the G794N 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 G794N would have decreased catalytic activity in comparison to the wild type. However, based on the results of the study, we created Michaelis-Menten graphs and Lineweaver-Burk plots to calculate the Km, Vmax, Kcat and catalytic efficiency, all of which surprisingly increased from wild-type to the mutant. We saw that there was an approximately ten-fold increase in the kcat of the mutant, indicating a significant increase in the rate of reaction for the mutant G794N. Hence, we concluded that the steric conformation and polarity of the 794 residue may be as important for enzyme function as its electronic charge. Moreover, the more catalytically efficient G794N mutant enzyme may be of biotechnological significance.

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Glik, Masha, Gallagher, James, and Chin, Logan, "Impact of Glycine to Asparagine Substitution on Catalytic Activity of E. coli β-galactosidase" (2021). Undergraduate Course-Based Research Poster Fair Collection. Brown Digital Repository. Brown University Library. https://doi.org/10.26300/58qg-k302

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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 Glycine to Asparagine Substitution on Catalytic Activity of E. coli β-galactosidase