2024-03-29T12:33:42Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1280502021-12-27T16:24:54Zcom_10261_79com_10261_1col_10261_332
DIGITAL.CSIC
author
Rocha-Martín, Javier
author
Vega, Daniel
author
Bolívar Bolívar, Juan Manuel
author
Godoy, César A.
author
Hidalgo, Aurelio
author
Berenguer, José
author
Guisán, José Manuel
author
López-Gallego, Fernando
funder
Fundación Ramón Areces
funder
Comunidad de Madrid
funder
Ministerio de Ciencia y Tecnología (España)
funder
Comunidad de Madrid
funder
Consejo Superior de Investigaciones Científicas (España)
funder
Ministerio de Ciencia e Innovación (España)
funder
Fundación Ramón Areces
2016-01-25T12:22:01Z
2016-01-25T12:22:01Z
2011
BMC Biotechnology 11 (2011)
http://hdl.handle.net/10261/128050
10.1186/1472-6750-11-101
http://dx.doi.org/10.13039/100012818http://dx.doi.org/10.13039/501100003339http://dx.doi.org/10.13039/100008054http://dx.doi.org/10.13039/501100004837http://dx.doi.org/10.13039/501100006280
22053761
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. © 2011 Rocha-Martín et al; licensee BioMed Central Ltd.
eng
openAccess
Dehydrogenase
Immobilization
NAD+, extremophiles
New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD +-recycling enzyme
artículo
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