PT - JOURNAL ARTICLE AU - Theo Peschke AU - Sabrina Gallus AU - Patrick Bitterwolf AU - Yong Hu AU - Claude Oelschlaeger AU - Norbert Willenbacher AU - Kersten S. Rabe AU - Christof M. Niemeyer TI - Self-assembling all-enzyme hydrogels for biocatalytic flow processes AID - 10.1101/240325 DP - 2017 Jan 01 TA - bioRxiv PG - 240325 4099 - http://biorxiv.org/content/early/2017/12/27/240325.short 4100 - http://biorxiv.org/content/early/2017/12/27/240325.full AB - We describe the construction of binary self-assembling all-enzyme hydrogels that are comprised entirely of two tetrameric globular enzymes, the stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH. The enzymes were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerise under physiological conditions into porous hydrogels. The biocatalytic gels were used for the highly stereoselective reduction of a prochiral diketone substrate where they showed the typical behaviour of the coupled kinetics of coenzyme regenerating reactions in the substrate channelling regime. They effectively sequestrate the NADPH cofactor even under continuous flow conditions. Owing to their sticky nature, the gels can be readily mounted in simple microfluidic reactors without the need for supportive membranes. The reactors revealed extraordinary high space-time yields with nearly quantitative conversion (>95%), excellent stereoselectivity (d.r. > 99:1), and total turnover numbers of the expensive cofactor NADP(H) that are amongst the highest values ever reported.