Thermal inactivation of xanthine oxidase from Arthrobacter M3: mechanism and the corresponding thermostabilization strategy.

The mechanism of thermal inactivation about xanthine oxidase (XOD) from Arthrobacter M3 was investigated. Results of reducing SDS-PAGE indicated that the inactivation of XOD was not related to the peptide degradation. Meanwhile, fluorimetry and circular dichroism spectroscopy suggested that XOD inactivation might be associated with the exposure of hydrophobic residues to surface and partial loss of secondary structure. Specific formation of soluble aggregates of XOD was detected by size exclusion chromatography. In addition, the thermal-dynamic analysis showed that the inactivation kinetics of XOD followed the first-order model. Therefore, trehalose (cosolute) and betaine (osmolyte) were accordingly employed to attenuate the inactivation of this enzyme. The results associated with these two reagents further confirmed that the loss of XOD activity was mainly due to the exposure of hydrophobic residues and formation of aggregation. Owing to the added trehalose and betaine, half-life could be significantly increased, and the inactivation rate constant (k) was detected as decreased.