Browsing by Subject "Enzyme kinetics"

Now showing 1 - 1 of 1

Open Access
An engineered genetic circuit for protein glycosylation in Escherichia coli
(2021-05) Bozkurt, Eray Ulaş
Protein glycosylation is one of the most crucial and common post-translational modifications. Glycosylation provides certain advantages to host organisms and extend the proteome beyond genetic material. After the discovery of bacterial glycosylation mechanisms and especially after its transfer into laboratory work-horse E. coli, studies utilizing this mechanism increased exponentially. It has been previously showed that utilizing N-Linked Glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. In this study, we utilized N-linked Glycosylation to glycosylate alkaline phosphatase (ALP) enzyme in E. coli and investigate the effects of glycosylation on an enzyme. Considering the glycosylation mechanism is highly dependent on the acceptor protein, ALP constructs carrying glycosylation tag at different locations of the gene has been created and glycosylation rates have been calculated. The most glycosylated construct has been selected for comparison with the native enzyme. Studies showed that glycosylated ALP performed better at optimal conditions. In order to extend the knowledge on the differences due to glycosylation, several conditions were applied. Both enzymes were tested at elevated temperatures for different incubation times, different pH conditions, protease treatment and under denaturing conditions. Also, secondary structure analysis was performed for each condition to elaborate on these differences. Experiments showed that glycosylated ALP performs remarkably better at all conditions tested. Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.