Molecular Enzyme Evolution

© Fraunhofer IME | Helga Schinkel

3D-modeling of enzyme mutants can help to understand changed activities; due to amino acid exhange in the mutant there is more space in the active center (catalytic triade in red; mutated amino acids in blue)

Enzymes catalyze specific chemical reactions under mild conditions and therefore have the potential to replace the chemical catalysts used in industry while offering further advantages such as greater specificity, lower toxicity and more environmental sustainability. However, natural enzymes are not always suitable for industrial processes and must be tailored for the required specifications. This can be achieved using protein-accelerated evolution, a process in which libraries of mutants are generated and screened for improved properties in iterative cycles.

The key element of directed evolution is the screening step because a suitable assay must be developed to highlight the property of interest that needs to be selected. We work with artificial compartments based on water-in-oil emulsions that act as pico-reaction vessels, allowing us to evaluate more than ten million enzyme variants in less than one hour. Each droplet contains one enzyme variant and the substrate needed for the synthesis of a fluorescent product using the catalytic activity of the enzyme. Droplets containing the most appropriate enzyme variants can be isolated by flow cytometry/florescence activated cell sorting or microfluidic devices.

We have developed successful screening systems based on flow cytometry and/or microfluidic devices for several enzymes classes, including glucose oxidases, cellulases, xylanases, chitinases, peroxidases and cellobiose dehydrogenases.

Selected Publications

Ostafe, R., Prodanovic, R., Nazor, J., Fischer, R., 2014a. Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase. Chemistry & Biology 21, 414–421.

Ostafe, R., Prodanovic, R., Ung, W.L., Weitz, D.A., Fischer, R., 2014b. A high-throughput cellulase screening system based on droplet microfluidics. Biomicrofluidics 8, 41102.

Ostafe, R., Prodanovic, R., Commandeur, U., Fischer, R., 2013. Flow cytometry-based ultra-high-throughput screening assay for cellulase activity. Analytical Biochemistry 435, 93–98.