Development of Multi-Oxidoreductase Reaction Technology for Value-added Chemical Processes

Abstract

Enzyme technologies are being developed as an eco-friendly and sustainable process. The research direction is enzyme engineering, enzyme immobilization process optimization, and flow biocatalysis. Among them, technology development for manipulating oxidoreductase is required because of their wide application range. A protein immobilization technology for process simplification, a reaction system for flow biocatalysis of multi-oxidoreductase, and gas conversion through a combination of multi-oxidoreductase were developed. First, protein immobilization has been widely used for laboratory experiments and industrial processes. Preparing a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. To achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein. Still, not endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilizing superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with minimal non-target proteins (target-selective immobilization). Second, Oxidoreductases are widely used in value-added chemical synthesis. Since many oxidoreductases require the consumption of expensive cofactors, multi-enzyme cascade reactions, including cofactor regeneration, have been developed. Although enzymes are frequently immobilized for industrial processes, immobilizing small molecule cofactors is challenging. We demonstrate that co-immobilizing a swing arm cofactor and multiple oxidoreductases can overcome these cofactor limitations. We chose a flexible protein, elastin-like polypeptide (ELP), fused to a strep-tag II as a genetically encoded swing arm. The Escherichia coli expressed, and purified swing arm was conjugated with NAD to generate a cofactor swing arm (ELP-NAD+). The cofactor swing arm was co-immobilized on a streptavidin resin with two oxidoreductases fused to a strep-tag II: glucose dehydrogenase (GDH) and mannitol dehydrogenase (MDH). We observed substantial D-mannitol production in the GDH, MDH, and ELP-NAD+ co-immobilized microreactor. Moreover, the product could be easily separated from the co-immobilized reactor, and the activity was retained when applied for repeated batch reactions (> seven cycles). These results demonstrate that the genetically encoded cofactor swing arm can be stably co-immobilized with multiple oxidoreductases on a solid support. It can maintain reactivity as a cofactor because of the flexible ELP swing arm. Third, Hydrogen gas obtained from cheap or sustainable sources has been investigated as an alternative to fossil fuels. Hydrogenase (H2ase) and formate dehydrogenase (FDH) can convert H2 and CO2 gases to formate, which can be conveniently stored and transported. However, developing an enzymatic process that converts H2 and CO2 obtained from cheap sources into formate is challenging because even a very small amount of O2 in cheap sources damages most H2ases and FDHs. To overcome this limitation, we investigated a pair of oxygen-tolerant H2ase and FDH. We achieved the cascade reaction between H2ase from Ralstonia eutropha H16 (ReSH) and FDH from Rhodobacter capsulatus (RcFDH) to convert H2 and CO2 to formate using in situ regeneration of NAD+/NADH in the presence of O2. Fourth, it is challenging to capture carbon dioxide (CO¬¬2), a major greenhouse gas in the atmosphere, due to its high chemical stability. One potential practical solution to eliminate CO2 is to convert CO2 into formate using hydrogen (H2) (CO2 hydrogenation), which can be accomplished with inexpensive hydrogen from sustainable sources. While industrial flue gas could provide an adequate source of hydrogen, a suitable catalyst is needed that can tolerate other gas components, such as carbon monoxide (CO) and oxygen (O2), potential inhibitors. Our proposed CO2 hydrogenation system uses the hydrogenase derived from Ralstonia eutropha H16 (ReSH) and formate dehydrogenase derived from Methylobacterium extorquens AM1 (MeFDH1). Both enzymes are tolerant to CO and O2, which are typical inhibitors of metalloenzymes found in flue gas. We have successfully demonstrated that combining ReSH- and MeFDH1-immobilized resins can convert H2 and CO2 in real flue gas to formate via a nicotinamide adenine dinucleotide-dependent cascade reaction.