Authors:

Dominic Cincetti-Gallegos

Date Created:

Not specified

Course Title:

Professor:

Not specified

About Paper:

This project characterizes the structural and functional significance of the mutation Q19L in the B-glucosidase B (BgIB) enzyme to add data concerning enzymatic efficiency and stability to the Design2Data (D2D) database, thus expanding the current knowledge on enzymes. We initially proposed that this mutation would decrease its enzymatic activity, expression, and thermal stability because of the significant increase in the energy score in Foldit and the change in the amino acid's polarity. The pET29b plasmid with the mutated gene was isolated and purified before it was sent for sequencing. The mutated plasmid was then transformed into BL21 (DE3) E. coli cells; and protein expression was induced. The mutant protein was then purified with chromatographic purification. SDS-PAGE and Western-Blot analyses were used to characterize the size and confirm the presence of the protein, respectively. Enzymatic and thermal-stability assays were employed to determine the efficiency and stability of both the mutant enzyme and wild type enzyme. The results indicate that the mutation Q19L enhances the catalytic efficiency significantly and thermal stability only slightly of BgIB. Our findings raise questions on how significantly the amino-acid structure of an enzyme affects its activity.

Source:

Loyola University Chicago

Topics:

No topics listed

Co-authors:

Dominic Cincetti-Gallegos