title: New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme.
abstract: BACKGROUND
  The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.
  
  
  RESULTS
  The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.
  
  
  CONCLUSIONS
  We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.
authors: Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Cantoblanco, Madrid, Spain. jmguisan@icp.csic.es
authors: Javier
authors: J
authors: Rocha-Martín
authors: Daniel
authors: D
authors: Vega
authors: Juan M
authors: JM
authors: Bolivar
authors: Cesar A
authors: CA
authors: Godoy
authors: Aurelio
authors: A
authors: Hidalgo
authors: José
authors: J
authors: Berenguer
authors: José M
authors: JM
authors: Guisán
authors: Fernando
authors: F
authors: López-Gallego
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010626:Phenylethyl Alcohol / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D016963:Thermus thermophilus / Q000201:enzymology*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004800:Enzymes, Immobilized; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D009536:Niacinamide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D020830:rac1 GTP-Binding Protein; D010626:Phenylethyl Alcohol; D010100:Oxygen