Self-Assembling All-Enzyme Hydrogels for Flow Biocatalysis

Abstract

Continuous flow biocatalysis is an emerging field of industrial biotechnology that uses enzymes immobilized in flow channels for the production of value-added chemicals. We describe the construction of self-assembling all-enzyme hydrogels that are comprised of two tetrameric enzymes. The stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerize under physiological conditions into porous hydrogels. Mounted in microfluidic reactors, the gels show excellent stereoselectivity with near quantitative conversion in the reduction of prochiral ketones along with high robustness under process and storage conditions. The gels function as compartment that retains intermediates thus enabling high total turnover numbers of the expensive cofactor NADP(H).

Keywords: enzymes; flow biocatalysis; immobilization; microreactors; stereoselective reactions.