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Open Access Research article

New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

Javier Rocha-Martín1, Daniel Vega2, Juan M Bolivar1, Cesar A Godoy1, Aurelio Hidalgo2, José Berenguer2, José M Guisán1* and Fernando López-Gallego1*

Author Affiliations

1 Departamento de Biocatálisis. Instituto de Catálisis y Petroleoquímica-CSIC. Campus UAM. Cantoblanco. 28049 Madrid, Spain

2 Centro de Biología Molecular Severo Ochoa. CSIC-UAM. Departamento de Biología Molecular. Campus UAM. Cantoblanco. 28049 Madrid, Spain

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BMC Biotechnology 2011, 11:101  doi:10.1186/1472-6750-11-101

Published: 3 November 2011

Additional files

Additional file 1:

Figure S1. Analysis SDS-PAGE of NOX purfication. SDS-PAGE (12%) gels obtained during the sequential purification of NOX. Lanes: 1) Molecular weight markers; 3) Crude extract; 5) Supernatant after heat treatment at 80°C for 45 minutes; 6) Supernatant after incubation in presence of PEI agarose for 30 minutes; 8) Supernatant after incubation in the presence of sulfate-dextran agarose for 1 h.

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Additional file 2:

Table S1. H2O2 formation during NAD+ reduction by NOX. The enzyme produces exclusively H2O2 as previously described Park et al. The fact that the recovery of H2O2 was sometimes less than 100% (70-80%), could be explained by the observation that the amount of H2O2 measured, was influenced by the time period between the NADH conversion and the actual measurement of H2O2. Apparently, the amount of H2O2 in the assay mixture slowly decreased, despite the absence of NADH, which was already completely converted at that moment.

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Additional file 3:

Figure S2. Alignment of NADH oxidase isolated from Thermus thermophilus HB27 and its counterpart isolated form Thermus thermophilus HB8. Sequence HB27* resulted from cloning and sequencing of PCR product amplified from genomic DNA of Thermus thermophilus HB27 that we have at our laboratory. The sequence HB8 was corresponding to gene bank accession number: CAA42707.1. Both sequences were aligned using ClustalW algorithm. (*) identical residues. (:) different residues, highlighted in grey.

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