Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans

Daisuke Sugimori, Yusaku Matsumoto, Yu Tomita, Kazutaka Murayama, Chiaki Ogino

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

Abstract

Sphingomyelinase C (SMC) of the actinomycete, Streptomyces griseocarneus 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 k cat 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.

Original languageEnglish
Pages (from-to)151-156
Number of pages6
JournalProtein Expression and Purification
Volume81
Issue number2
DOIs
Publication statusPublished - 2012 Feb 1

Keywords

  • Enzymatic activity enhancement by MgCl
  • Expression
  • Kinetics
  • Sphingomyelinase C
  • Streptomyces

ASJC Scopus subject areas

  • Biotechnology

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