Microreactor technology for continuous enzymatic reactions catalyzed by C-C-bond forming enzymes

acronym: MicroTechEnz

Results of MicroTechEnz
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Project coordinator

- Prof. Ðurða Vasiæ-Raèki - University of Zagreb - Croatia
Project leaders
- Prof. Martina Pohl - Forschungszentrum Jülich GmbH - Germany
- Prof. Pere Clapés - Instituto de Química Avanzada de Catalunya
  (IQAC) / Consejo Superior de Investigaciones Científicas
  (CSISC) - Spain
- Mr Sergi Pumarola - Bioglane S.L.N.E. - Spain
- Mr W. Bolt (subcontractor) - Micronit Microfluidics BV -
  The Netherlands

The present project deals with the evaluation of microreactor technology for enzymatic carboligase reactions using thiamin diphosphate (ThDP)-dependent enzymes (TDEs)and D-fructose-6-phosphate aldolase from E.coli (FSA).The main goal for the TDEs is to develop micro-reactor technology as a screening tool to identify the appropriate enzyme for a desired carboligation reaction of two aldehydes yielding chiral 2-hydroxy ketones. Here, the prediction of the process relevant data concerning e.g. optimal substrate concentration, selectivity, enzyme stability and productivity with very small amounts of enzymes and chemicals will be tested using micro-reactor technology in comparison to conventional lab scale reactors. The main advantage of FSA is that it dispenses with the need for laborious preparation of a sensitive phosphorylated reagent, dihydroxyacetone phosphate (DHAP), essential for DHAP-dependent aldolase, and it is currently used for the large scale production of D-fagomine. Notwithstanding the obvious advantages, some emerging issues limit its broad synthetic applicability while others can be significantly improved making them attractive from industrial point of view. Among them, are the following:
A) Concerning the acceptor substrate selectivity, substrate inhibition, and the thermodynamic limitations of the catalyzed reactions.
B) Improving performance by cascade reactions with in situ aldehyde generation.
C) Improving molecular diversity by cascade two-aldol additions with in situ product formation. These issues can be effectively optimized in micro-reactors and/or in combination with protein engineering.

The project would bring new insights into green chemical syntheses reactions which are of vital interest to the field of high technologies, such as industrial biotechnology and approve a new concept for the production of enantiomerically pure diols and iminocyclitols. Technology transfer to end users, who are involved in the project, will be realized. Micro-reactors integrating oxidation, aldol reaction, enzyme separation and downstream processing are of utmost importance to develop a competitive process and the present project will provide technological solutions and will represent the first real example of industrial biotechnology development of an active process using aldolases.