Trehalose-mediated thermal stabilization of glucose oxidase from Aspergillus niger.
Thermal inactivation and enzyme kinetics of glucose oxidase (a FAD dependent enzyme) were studied in the absence and presence of trehalose. The inactivation rate constant decreased by up to 50% at temperatures between 50 and 70 degrees C in the presence of 0.6M trehalose; as a consequence the glucose oxidase half-life increased. Intrinsic fluorescence spectra showed a maximum center of spectral mass (CSM) red shift of 6.5nm. Therefore, major structural changes seem to be related to glucose oxidase thermal inactivation. Trehalose decreased the rate constant for unfolding as monitored by CSM red shift kinetics indicating that this disaccharide favors the most compact folded state. The E(a) for unfolding was increased from 204 to 221kJ mol(-1). It is proposed that FAD dissociation is preceded by the exposition of hydrophobic regions, while the presence of trehalose was able to hinder the release of FAD. Enzyme kinetics analysis showed that trehalose does not affect V(max) but instead decreases K(m); as a result enzyme efficiency was increased. The stabilizing effect of trehalose in a cofactor-dependent enzyme has not been tested to date. In addition, glucose oxidase has an enormous commercial importance and therefore, the use of trehalose to stabilize glucose oxidase in its multiple applications seems to be promising.
Paz-Alfaro
Karina J
KJ
Area Académica de Nutrición, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Hidalgo, Mexico.
Ruiz-Granados
Yadira G
YG
Uribe-Carvajal
Salvador
S
Sampedro
José G
JG
D001234:Aspergillus niger / Q000201:enzymology*; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005949:Glucose Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014199:Trehalose / Q000378:metabolism*
D005182:Flavin-Adenine Dinucleotide; D014199:Trehalose; D005949:Glucose Oxidase