Electro-enzymatic viologen-mediated substrate reduction using pentaerythritol tetranitrate reductase and a parallel, segmented fluid flow system

Many redox enzymes require expensive reduced cofactors like NAD(P)H which need to be recycled during catalysis, presenting a major cost and technical barrier to industrial exploitation. An electrochemical biphasic microfluidic setup is presented here, in which these cofactors are replaced by a mediator (methyl viologen) that acts by feeding electrons into the active site of the enzyme pentaerythritol tetranitrate reductase (PETNR). In this microfluidic recirculation setup, both enzyme and mediator remain in the reactor for reuse, allowing easy product recovery. System optimisation studies were performed using 2-cyclohexen-1-one as a model substrate prior to the investigation of a variety of different substrates whose reduction rates were determined to be 15–70% of those obtained when NADPH was used as sole electron donor. Additional data obtained with a thermophilic ‘ene’ reductase (TOYE) support the potential universality of this device for possible industrial applications.

[1]  N. Scrutton,et al.  Biocatalytic Reductions and Chemical Versatility of the Old Yellow Enzyme Family of Flavoprotein Oxidoreductases , 2010 .

[2]  V. Massey,et al.  Old yellow enzyme: stepwise reduction of nitro-olefins and catalysis of aci-nitro tautomerization. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Remko M Boom,et al.  Comparison of two‐phase lipase‐catalyzed esterification on micro and bench scale , 2008, Biotechnology and bioengineering.

[4]  T. Tzedakis,et al.  Electrochemical microreactor for chiral syntheses using the cofactor NADH , 2008 .

[5]  Bernd Nidetzky,et al.  Biotransformations in microstructured reactors: more than flowing with the stream? , 2011, Trends in biotechnology.

[6]  David W. M. Marr,et al.  Hydrodynamic focusing for vacuum-pumped microfluidics , 2005 .

[7]  J. Collin,et al.  Electrochemical regeneration of nicotinamide cofactor using a polypyrrole rhodium bis-terpyridine modified electrode , 1993 .

[8]  N. Scrutton,et al.  Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation. , 2009, Advanced synthesis & catalysis.

[9]  W. Stręk,et al.  Electroreduction of methyl viologen in methanol and silicate thin films prepared by the sol–gel method , 2003 .

[10]  N. Scrutton,et al.  Biocatalysis with Thermostable Enzymes: Structure and Properties of a Thermophilic ‘ene’‐Reductase related to Old Yellow Enzyme , 2010, Chembiochem : a European journal of chemical biology.

[11]  J. Wiegel,et al.  Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic anaerobe from Yellowstone National Park. , 2007, International journal of systematic and evolutionary microbiology.

[12]  N. Scrutton,et al.  A surprising observation that oxygen can affect the product enantiopurity of an enzyme‐catalysed reaction , 2012, The FEBS journal.

[13]  Christopher M.A. Brett,et al.  A glucose biosensor using methyl viologen redox mediator on carbon film electrodes , 2005 .

[14]  Seong Kee Yoon,et al.  Laminar flow-based electrochemical microreactor for efficient regeneration of nicotinamide cofactors for biocatalysis. , 2005, Journal of the American Chemical Society.

[15]  M. Fujita,et al.  Electrochemical study of reversible hydrogenase reaction of Desulfovibrio vulgaris cells with methyl viologen as an electron carrier. , 1999, Analytical chemistry.

[16]  A. Fry,et al.  Electroenzymatic synthesis (regeneration of NADH coenzyme) : use of nafion ion exchange films for immobilization of enzyme and redox mediator , 1994 .

[17]  David R. Emerson,et al.  Numerical and experimental study of a droplet-based PCR chip , 2007 .

[18]  P. Boivin,et al.  Rapid electrocatalytic procedure for hydrogenase kinetic determination in the H2 evolution direction. , 1986, Biochemical and biophysical research communications.

[19]  Andreas Schmid,et al.  Miniaturizing Biocatalysis: Enzyme‐Catalyzed Reactions in an Aqueous/Organic Segmented Flow Capillary Microreactor , 2011 .

[20]  Asterios Gavriilidis,et al.  Flow Distribution in Different Microreactors Scale-Out Geometries and the Effect on Manufacturing Tolerances and Channel Blocking , 2004 .

[21]  Stephan Mohr,et al.  Continuous two-phase flow miniaturised bioreactor for monitoring anaerobic biocatalysis by pentaerythritol tetranitrate reductase. , 2010, Lab on a chip.

[22]  N. Scrutton,et al.  Biotransformation of Explosives by the Old Yellow Enzyme Family of Flavoproteins , 2004, Applied and Environmental Microbiology.

[23]  A. Abate,et al.  Syringe-vacuum microfluidics: A portable technique to create monodisperse emulsions. , 2011, Biomicrofluidics.

[24]  Frank Hollmann,et al.  Electrochemical Regeneration of Oxidoreductases for Cell-free Biocatalytic Redox Reactions , 2004 .

[25]  N. Scrutton,et al.  Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site‐Saturated Libraries , 2010, Chembiochem : a European journal of chemical biology.

[26]  W. Metcalf,et al.  Phosphite dehydrogenase: a versatile cofactor-regeneration enzyme. , 2002, Angewandte Chemie.

[27]  J. Burnett,et al.  Electrochemical Reduction of Diphosphopyridine Nucleotide , 1965 .

[28]  Dietmar Haltrich,et al.  Continuous enzymatic regeneration of redox mediators used in biotransformation reactions employing flavoproteins , 2001 .

[29]  P. Adlercreutz.  Cofactor regeneration in biocatalysis in organic media , 1996 .