Imine Reductase Based All-Enzyme Hydrogel with Intrinsic Cofactor Regeneration for Flow Biocatalysis

Abstract

All-enzyme hydrogels are biocatalytic materials, with which various enzymes can be immobilized in microreactors in a simple, mild, and efficient manner to be used for continuous flow processes. Here we present the construction and application of a cofactor regenerating hydrogel based on the imine reductase GF3546 from Streptomyces sp. combined with the cofactor regenerating glucose-1-dehydrogenase from Bacillus subtilis. The resulting hydrogel materials were characterized in terms of binding kinetics and viscoelastic properties. The materials were formed by rapid covalent crosslinking in less than 5 min, and they showed a typical mesh size of 67 ± 2 nm. The gels were applied for continuous flow biocatalysis. In a microfluidic reactor setup, the hydrogels showed excellent conversions of imines to amines for up to 40 h in continuous flow mode. Variation of flow rates led to a process where the gels showed a maximum space-time-yield of 150 g·(L·day)−1 at 100 μL/min.

Rapid Model-Based Optimization of a Two-Enzyme System for Continuous Reductive Amination in Flow

“…There are many documented benefits of flow to organic synthesis, − but more specific to biocatalysis is that immobilized enzymes can be used in continuous packed-bed reactors. Immobilization both enhances the stability of an enzyme and permits recyclability, substantially improving the economics of a biocatalytic process. − While there are a number of examples of conducting biocatalysis in flow, − the optimization of these reactions, particularly where multiple enzymes are coimmobilized, remains challenging.…”

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

“…146 In addition, and in contrast to conventional enzyme crosslinking methods, mild fusion conditions are required to obtain porous hydrogel networks. 147 First results showed high conversion rates and stereoselectivities as well as high space-time yields. 121,147 In addition, the in situ generation of pure enzyme hydrogels easily enables an increase in protein loading for microfluidic bioreactors.…”

“…147 First results showed high conversion rates and stereoselectivities as well as high space-time yields. 121,147 In addition, the in situ generation of pure enzyme hydrogels easily enables an increase in protein loading for microfluidic bioreactors. It is also Fig.…”

Streamlining Design, Engineering, and Applications of Enzymes for Sustainable Biocatalysis

“…A third affinity system involves fusion proteins with avidin–enzyme fusion proteins, where the avidin region binds to biotin activated surfaces . More sophisticated techniques include the Spytag/Spycatcher system, , which permits spontaneous formation of isopeptide bonds to immobilize proteins. The Spycatcher domain can be incorporated into a self-assembling bacterial microcompartment protein scaffold that will covalently immobilize SpyTagged enzymes such as an alcohol dehydrogenase …”