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

Abstract:

: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 MgCl(2), and the maximum activity (542±25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl(2). 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 (k(cat)), K(m) and k(cat)/K(m) values for SM were 346 s(-1), 0.458 mM, and 756 mM(-1)s(-1), respectively, in the presence of 1M MgCl(2). The k(cat) was strongly influenced by the MgCl(2) concentration. By contrast, the K(m) value was independent of the MgCl(2) concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl(2) did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl(2) was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.

journal_name

Protein Expr Purif

authors

Sugimori D,Matsumoto Y,Tomita Y,Murayama K,Ogino C

doi

10.1016/j.pep.2011.10.004

subject

Has Abstract

pub_date

2012-02-01 00:00:00

pages

151-6

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(11)00277-4

journal_volume

81

pub_type

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