Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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Sphingomyelinase C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a single band of 38kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF MS was 32,102Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found at pH 9.0 and 50-55℃, and the enzyme was stable from pH 5 to 10 and up to 37℃. The SMC activity requires Mg^<2+> or Mn^<2+> and the order of potency to enhance the activity was Zn^<2+>≥Mn^<2+>>Cu^<2+>≥Fe^<2+>. Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were determined as H_2N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020bp encoding a signal peptide of 42 amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125Da. The conserved region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.