Search Results (3)

Tetrahydrofolate (THF), the biologically active form of folate, serves as a crucial carrier of one-carbon units essential for synthesizing cellular components such as amino acids and purine nucleotides in vivo. It also acts as an important precursor for the production of pharmaceuticals, including folinate and L-5-methyltetrahydrofolate (L-5-MTHF). In this study, we developed an efficient enzyme cascade system for the production tetrahydrofolate from folate, incorporating NADPH recycling, and explored its application in the synthesis of L-5-MTHF, a derivative of tetrahydrofolate. To achieve this, we first screened dihydrofolate reductases (DHFRs) from various organisms, identifying SmDHFR from Serratia marcescens as the enzyme with the highest catalytic activity. We then conducted a comparative analysis of formate dehydrogenases (FDHs) from different sources, successfully establishing an NADPH recycling system. To further enhance biocatalytic efficiency, we optimized key reaction parameters, including temperature, pH, enzyme ratio, and substrate concentration. To address the challenge of pH mismatch in dual-enzyme reactions, we employed an enzymatic microenvironment regulation strategy. This involved covalently conjugating SmDHFR with a superfolder green fluorescent protein mutant carrying 30 surface negative charges (−30sfGFP), using the SpyCatcher/SpyTag system. This modification resulted in a 2.16-fold increase in tetrahydrofolate production, achieving a final yield of 4223.4 µM. Finally, we extended the application of this tetrahydrofolate synthesis system to establish an enzyme cascade for L-5-MTHF production with NADH recycling. By incorporating methylenetetrahydrofolate reductase (MTHFR), we successfully produced 389.8 μM of L-5-MTHF from folate and formaldehyde. This work provides a novel and efficient pathway for the biosynthesis of L-5-MTHF and highlights the potential of enzyme cascade systems in the production of tetrahydrofolate-derived compounds. Full article

Multi-enzyme cascade catalysis involved three types of dehydrogenase enzymes, namely, formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH), alcohol dehydrogenase (ADH), and an equimolar electron donor, nicotinamide adenine dinucleotide (NADH), assisting the reaction is an interesting pathway to reduce thermodynamically stable molecules of CO₂ from the atmosphere. The biocatalytic sequence is interesting because it operates under mild reaction conditions (low temperature and pressure) and all the enzymes are highly selective, which allows the reaction to produce three basic chemicals (formic acid, formaldehyde, and methanol) in just one pot. There are various challenges, however, in applying the enzymatic conversion of CO₂, namely, to obtain high productivity, increase reusability of the enzymes and cofactors, and to design a simple, facile, and efficient reactor setup that will sustain the multi-enzymatic cascade catalysis. This review reports on enzyme-aided reactor systems that support the reduction of CO₂ to methanol. Such systems include enzyme membrane reactors, electrochemical cells, and photocatalytic reactor systems. Existing reactor setups are described, product yields and biocatalytic productivities are evaluated, and effective enzyme immobilization methods are discussed. Full article

Formaldehyde is utilized for the preservation of materials due to its strong bactericidal effects. As formaldehyde is also a harmful substance that causes health hazards, the quantitative monitoring of formaldehyde in natural and living environments is desirable. For the rapid and easy detection of formaldehyde, in this study we applied an organic field-effect transistor (OFET)-based sensor that can function as a potentiometric device for electrochemical measurements. A polyion-complex gel of formaldehyde dehydrogenase (FDH) and chitosan (CT) was constructed on a gold electrode. When the FDH/CT gel-coated electrode was connected to an OFET device it could detect formaldehyde in an aqueous solution, in which the amino groups of chitosan would protonate during the enzymatic reaction. The limit of detection was calculated to be 3.1 µM (93 ppb), demonstrating the applicability of the film-type OFET sensor to environmental monitoring. Full article