(169d) Application of the Consensus Concept for Increased Thermostability of Enzymes

Vazquez-Figueroa, E., School of Chemical and Biomolecular Engineering, Georgia Institute of Technology
Chaparro-Riggers, J., Georgia Institute of Technology
Polizzi, K. M., School of Chemical and Biomolecular Engineering, Georgia Institute of Technology
Bommarius, A. S., Georgia Institute of Technology

The main factor preventing the widespread adoption of biocatalysts is their lack of stability under processing conditions at elevated temperatures, which are often used for their beneficial effects on reaction rates, reactant solubility, and to reduce the risk of microbial contamination. Therefore, enzymes with higher thermostability are necessary to create economically viable biocatalytic processes.

The thermostabilization of Penicillin G acylase is a difficult problem due to the large size of the protein and its complex maturation process. We developed a data-driven protein design method that requires fewer homologous sequences than the traditional consensus approach and utilizes structural information to limit the number of variants created. Approximately 50% of our 21 single-mutants were found experimentally to be more thermostable than the wild-type PGA, a significant increase in success rate when compared to other traditional techniques such as directed evolution.

For effective cofactor regeneration, we aim to increase the temperature stability of glucose dehydrogenase (GDH) via the consensus concept. GDH is a homotetramer capable of accepting NAD+ or NADP+ with high specific activity, making it an attractive target. Currently, we have successfully cloned, expressed, and purified the GDH encoding gene from Bacillus subtilis and Geobacillus stearothermophilus in Escherichia coli. Using the consensus approach we have generated 15 variants with potentially increased thermostability.

In this presentation we discuss the applicability of the consensus method to the development of a thermostable PGA and GDH, thermostability results on the generated variants, and progress to date.