New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme
Entity
UAM. Departamento de Biología MolecularPublisher
BioMed Central Ltd.Date
2011-11-03Citation
10.1186/1472-6750-11-101
BMC Biotechnology 11 (2011): 101
ISSN
1472-6750DOI
10.1186/1472-6750-11-101Funded by
Financial support from Madrid Region council CAM (grants S0505/PPQ/0344), CSIC (JAE-doc 108) and the Spanish Ministry of Science (Programa Ramón y Cajal and grants BIO-2008-01481, BIO2010-18875 and CTQ2009-07568) is gratefully acknowledged. An institutional grant from the Fundación Ramón Areces to the CBMSO is also acknowledged.Editor's Version
http://dx.doi.org/10.1186/1472-6750-11-101Subjects
Dehydrogenase; Extremophiles; Immobilization; Nad+; Biología y Biomedicina / BiologíaNote
Rocha-Martín et al.: New biotechnological perspectives of a NADH oxidase variant from Thermus thermophiles HB27 as NAD+-recycling enzyme. BMC Biotechnology 2011 11:101. The electronic version of this article is the complete one and can be found online at http://www.biomedcentral.com/1472-6750/11/101Rights
© 2011 Rocha-Martín et al; licensee BioMed Central Ltd.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.Conclusion: 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
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Google Scholar:Rocha-Martín, Javier
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Vega, Daniel
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Bolívar, Juan Manuel
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Godoy, César A.
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Hidalgo Huertas, Aurelio
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Berenguer Carlos, José
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Guisan, J. M.
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López-Gallego, Fernando
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