Abstract

The objective of this proposal is to obtain novel functional biocatalysts for regio- and enantio-selective oxidation working in a 'reactor plugged to a wall socket'. A central part is the isolation of the responsible part of monooxygenases and the combination with an electrochemical regeneration system. Our aim is to use sophisticated mediator technology to develop biocatalysts, which, in an industrial setting, are competitive to chemical catalysts used in hydroxylation. Together with an integration of mechanistic information of the enzymes and genetic engineering techniques, a large number of mutants prepared by directed evolution would be analysed by novel massive parallel screening methods. Based on the developments a basically new electro-enzymatic reactor will be constructed for chemical bulk synthesis of oxygenated fine chemicals.

Objectives

The main objective of this proposal is to obtain novel functional biocatalysts for regio- and enantio-selective oxidation working in a 'reactor plugged to a wall socket'. In their natural environment in the "cell factory", hydroxylases function in combination with a variety of other biomolecules. The isolation of the responsible part of the oxidation cascade and the combination with an electrochemical regeneration system is the central part of the proposal. We will develop highly efficient biocatatalysts with programmable properties for new bioprocesses and will produce a deep insight into the mechanism of biological oxidations. Our aim is to use new mediator technology to develop biocatalysts, which are competitive with chemical catalysts used in hydroxylation. Together with an integration of mechanistic information of the enzymes and genetic engineering techniques a large number of mutants prepared by directed evolution would be analysed by novel massive parallel screening methods.

Description of the work

The work on the improvement of monooxygenases for industrial applications can be divided in two main topics:

• The development of highly efficient biomolecules by directed molecular evolution and rational design: This includes the development of new assay systems for high throughput screening, suitable for automation. The synthesis of appropriate compounds for selective colorimetric assays and the investigation of biosensors based on micro array technology for direct detection of selective hydroxylation catalysts on electrode surfaces. Homology modelling of wild type and mutant enzymes as well as molecular dynamics simulation of enzyme-substrate interactions will be examined to understand catalytic mechanism and enzyme selectivity. A high throughput screening on an automated robotic system for new selective, stable and electrochemical regenerable monooxygenase variants will do.
• The investigation and application of the electron transfer processes from electrode surfaces to the enzyme active site: This includes the construction of strains for the production of wild type and enzyme variants in milligram to gram scale. The isolation of the catalytic-responsible part and the purification of these proteins for the immobilisation on electrode surfaces and application purposes. The development of a direct and indirect electrochemical regeneration system for monooxygenase variants and transfer to electro-enzyme reactor for continuous synthesis in preparative scale.

• Strains for monooxygenase production and monooxygenase mutation will developed suitable for the generation of grams of enzymes and high throughput screening, respectively.
• Direct regeneration systems for monooxygenase on electrodes will be established.
• A new HTS system using micro arrays will lead to an improved monooxygenase for industrial biotransformation.
• The (in)direct regeneration system will run with improved monooxygenase in the electro-enzyme reactor for synthesis of fine chemicals.

Contacts

Coordinator

• Bernhard HAUER / BASF AG GERMANY

Participant

• Institut für Technische Biochemie ITB GERMANY
• Andreas SCHMID / Institute of Biotechnology SWITZERLAND
• H. Allen O. Hill / University of Oxford Inorganic Chemistry Laboratory / UNITED KINGDOM
• Alain BERGEL / Université Paul Sabatier Laboratoire de génie Chimique - CNRS / FRANCE
• N.P.E. VERMEULEN / Vrije Universiteit LACDR - Pharmacochemistry / NETHERLANDS