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bioRxiv · 10.1101/2021.11.15.468597

Protein Engineering with A Glycosylation Circuit Enables Improved Enzyme Characteristics

Bozkurt, E.·Cagil, I.·Kehribar, E.·Isilak, M.·Seker, U. O. S.
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Abstract

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins, making it an interesting for many biotechnology applications. The discovery of bacterial glycosylation mechanisms, opened a new perspective and transfer of C.jejuni N-linked glycosylation into laboratory work-horse E. coli increased research pace in the field 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. We investigated the performance of glycosylated ALP in terms of its thermostability, proteolytic stability, tolerance to suboptimal pH and under denaturing conditions. Studies showed that glycosylated ALP performed remarkably better at optimal and harsh conditions Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.

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Bozkurt, E., Cagil, I., Kehribar, E., Isilak, M., Seker, U. O. S.. 2021-11-15. Protein Engineering with A Glycosylation Circuit Enables Improved Enzyme Characteristics. https://doi.org/10.1101/2021.11.15.468597

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