| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2020693 | Protein Expression and Purification | 2012 | 6 Pages |
Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550–950 U/mg) and was obtained in high yields (approximately 120 mg/L of culture). SMC activity was enhanced by MgCl2, and the maximum activity (542 ± 25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl2. Dynamic light scattering analysis proved that the highest specific SMC activity was obtained with the smallest mixed micelles of sphingomyelin (SM) and Triton X-100. The turnover rate (kcat), Km and kcat/Km values for SM were 346 s−1, 0.458 mM, and 756 mM−1 s−1, respectively, in the presence of 1 M MgCl2. The kcat was strongly influenced by the MgCl2 concentration. By contrast, the Km value was independent of the MgCl2 concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl2 did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl2 was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.
► Sphingomyelinase C of Streptomycesgriseocarneus was recombinantly expressed using Streptomyces lividans. ► It (35.2 mg, 952 U/mg) was purified from the culture supernatant. ► The Km and kcat for sphingomyelin were 0.458 mM and 346 s−1. ► At higher MgCl2 concentrations, the sphingomyelinase C activity increased. ► This was caused by the increased specific surface area of the substrate micelles.
