ABSTRACT
We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H2. Coupling this system to an ene reductase, we show quantitative conversion of N-ethylmaleimide to N-ethylsuccinimide. The biocatalyst system retained >50% activity after 7 h.
Subject(s)
Hydrogenase , Ethylmaleimide , Hydrogen , Hydrogenase/metabolism , NAD/metabolism , Niacinamide , Oxidation-Reduction , Oxidoreductases/metabolism , SuccinimidesABSTRACT
We describe the implementation of a system of immobilised enzymes for H2-driven NADH recycling coupled to a selective biotransformation to enable H2-driven biocatalysis in flow. This approach represents a platform that can be optimised for a wide range of hydrogenation steps and is shown here for enantioselective ketone reduction and reductive amination.
Subject(s)
Biocatalysis , Enzymes, Immobilized/metabolism , Hydrogen/metabolism , Hydrogenase/metabolism , Nanotubes, Carbon/chemistry , Oxidoreductases/metabolism , Amination , Hydrogen/chemistry , Hydrogenation , Ketones/chemistry , Ketones/metabolism , Oxidation-ReductionABSTRACT
We describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10-1000 s(-1), and therefore, fast experimental triggers are needed in order to study subturnover kinetics and identify the involvement of transient species important to their catalytic mechanism. In an electrochemical experiment, this equates to the use of microelectrodes to lower the electrochemical cell constant and enable changes in potential to be applied very rapidly. We use a biological cofactor, flavin mononucleotide, to demonstrate the power of synchrotron infrared microspectroscopy relative to conventional infrared methods and show that vibrational spectra with good signal-to-noise ratios can be collected for adsorbed species with low surface coverages on microelectrodes with a geometric area of 25 × 25 µm(2). We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononucleotide active site of a flavoenzyme. The method we describe will allow time-resolved spectroscopic studies of chemical and structural changes at redox sites within a variety of proteins under precise electrochemical control.
Subject(s)
Electrochemical Techniques , Flavin Mononucleotide/chemistry , Spectrophotometry, Infrared , Biocatalysis , Catalytic Domain , Electrodes , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Oxidation-Reduction , Quinone Reductases/chemistry , Quinone Reductases/metabolism , Soot/chemistry , SynchrotronsABSTRACT
An in vivo biotransformation system is presented that affords the hydroxylation of n-octane to 1-octanol on the basis of NADH-dependent CYP153A monooxygenase and NAD(+)-reducing hydrogenase heterologously synthesized in a bacterial host. The hydrogenase sustains H2-driven NADH cofactor regeneration even in the presence of O2, the co-substrate of monooxygenase.
