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PublicationRSC Advances
Year2015
Volume5
Pages53047 - 53053.
International

OPTIMIZATION AND CHARACTERIZATION OF CLEAS OF THE VERY THERMOSTABLE DIMERIC PEROXIDASE FROM Roystonea regia

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Alba Moralesa,+,  Oveimar Barbosab,+, Nazzoly Ruedaa,c, Zayda Fonsecaa, Rodrigo Torresa,d,
Rafael C. Rodriguese, Claudia Ortizf *, Roberto Fernandez-Lafuentec,*
 
a Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
b Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia
c Departamento de Biocatálisis. Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid. Spain.
dCurrent address: Laboratorio de Biotecnología, Instituto Colombiano del Petróleo-Ecopetrol, Piedecuesta, Bucaramanga, Colombia.
e  Biocatalysis and Enzyme Technology Lab, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, Porto Alegre, RS, Brazil.
fEscuela de Bacteriología y Laboratorio Clínico, Universidad Industrial de Santander, Bucaramanga, Colombia.



This paper describes the optimization of the immobilization of the peroxidase from Roystonea regia (RPTP) using the technique of the crosslinking enzyme aggregates. The immobilization was optimized using response surface methodology and after evaluating three different precipitants, ethanol was finally selected. Three variables were analyzed, glutaraldehyde concentration, precipitant concentration and time before collecting the RPTP-CLEAs. The immobilization yield was around 75%. The activity of the RPTP-CLEA was very high, this was even more patent at pH 3, where the free RPTP was fully inactive and the RPTP-CLEA maintained 40% of the maximum activity. In stress inactivations, the RPTP-CLEA maintained the very high thermostability that presented the free enzyme at 90ºC and pH 7 (half-live of 50 min). At pH 3, the free enzyme suffered subunit dissociation as a first step of the inactivation, this is not possible using RPTP-CLEAs and provided a very high thermostabilization (that depends on the enzyme concentration). The stability of the enzyme in the presence of hydrogen peroxide is good at low concentrations of this reagent (e.g., 10 mM), but if the concentration is higher (e.g., 300 mM), the enzyme stability drops. The immobilization provides an improved stability in the presence of this oxidant, but the values reached may not be high enough for some applications. The RPTP-CLEAs may be used for the decoloration of methyl orange solutions using 5 mM of hydrogen peroxide for 4 cycles (4 h each cycle) without apparent decrease in activity (but only degrading around 50% of the substrate). Using 225 mM of this oxidative reagent, the activity slowly decreased after each cycle (but enabling the full destruction of the colorant). This immobilized enzyme may be used even at pH 3 and 225 mM hydrogen peroxide, conditions where the free enzyme is completely inactive.
Keywords:crosslinked enzyme aggregates optimization, multimeric enzyme stabilization, hydrogen peroxide inactivation, peroxidase immobilization, response surface methodology
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