On the Enzymatic Properties and Deactivation Mechanism of Sarcosine Oxidase

Abstract:

The sarcosine oxidase（SOX） gene from Bacillus sp. was expressed in E. coli，and the properties and deactivation mechanism were further analyzed. After transformation of the cloned DNA into host E. coli BL21（DE3），the SOX gene was induced by IPTG for 8 h，a protein band of 43 kDa appeared in SDS-PAGE electrophoresis. From the crude enzyme solution，SOX in high purity was obtained through affinitive separation and purification. The analytic results of enzymatic properties showed that its relative molecular mass was 43.8 kDa， and its optimum reaction temperature and pH value were 40 ℃ and 8.0，respectively. Moreover，20 mmol/L Mn2+ exhibited an obvious activation effects on recombinant SOX. The kinetic parameters of Km and Vmax were determined as 141.6 mmol/L and 0.115 mmol/（L?min） when sarcosine used as a substrate. With the SDS-PAGE gel electrophoresis，fluorescence and circular dichroism spectroscopy，the deactivation mechanism of SOX was further studied. At constant temperature 37 ℃，the internal hydrophobic group of SOX molecule was gradually exposed and its secondary structure was destroyed along with the prolonged storage time，leading to the denaturation of SOX protein and the lowering down of its activity. The above results provide a theoretical basis for improving SOX stability in future.