Project Title: Knowledge-based redesign of a flavin-containing monooxygenase

Supervisor: Prof. Marco Fraaije, University of Groningen, The Netherlands

Objectives: By directed evolution approaches, the biocatalytic potential of a flavin-containing monooxygenase will be expanded. This will be achieved by generating and screening knowledge-based focused enzyme mutant libraries. By combining beneficial mutations, potent enzymes will be engineered for performing conversions of indole and related compounds. The mutant enzymes will be subjected to detailed studies in order to understand the effects of the mutations on the thermo- and operational stability, kinetic parameters and regio- and enantioselectivity.

Methodology: The work will involve various molecular biology and biochemical techniques. For the enzyme engineering work, Escherichia coli will be used as host. Mutant libraries will be prepared and screened, and the identified mutant enzymes will be purified and characterized. Enzyme characterization will include: kinetic analyses (spectrophotometric assays for steady-state kinetics, stopped-flow studies for pre-steady state kinetics), stability analyses (thermostability and solvent tolerance), and crystal structures will be determined in collaboration with the ESR in the group of Prof. Andrea Mattevi (protein crystallography). The generated enzymes will also be evaluated as biocatalyst (e.g. performing bioconversions with GC/LC analyses, optimizing biocatalytic process and conditions) in close collaboration with the ESR at ISKO.

Expected Results: (i) High quality enzyme mutant libraries and selection of improved enzyme mutants; (ii) full characterisation of the tailor-made enzymes by biophysical, kinetic and chemical analyses.