Expression, Purification and Characterization of Neutral Sphingomyelinase

• Main Authors: Shih-Chiao Tseng, 曾詩喬
• Other Authors: Shyh-Yu Shaw
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/08814647228293562196

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 90 === At the epicenter of the sphingomyelin-cell signaling pathway is a family of phospholipase called sphingomyelinase. These enzymes induce a variety of cell regulatory phenomenon such as programmed cell death (apoptosis), cell differentiation, cell proliferation, and sterol homeostasis. Sphingomyelinase may be useful for additional studies of apoptosis in experimental animals. We have identified a neutral sphingomyelinase from Bacillus subtilis IAM1208. The enzyme, with a molecular mass of 34.3 kDa, has 98.6% amino acid sequence identity with sphingomyelinase from Bacillus cereus IAM 1208. When N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified neutral-sphingomyelinase exhibited a Km of 11.34μM and a Vmax of 10.225 nmol /min/10μg of protein at 37℃, pH 7.4. The pH and ion effect on N-SMase activity was examined. The optimal pH of N-SMase was at pH 7.4 .The enzyme activity was inhibited by Ca2+ but was activated by Mg2+. The effect of bivalent cations on the activity and Tryptophan fluorescence spectra of N-SMase were also examined. In the Ca2+-dependence curve of tryptophan fluorescence intensity, one distinct transition was observed between 10-6 to 10-8M, In the Mg2+-dependence curve of fluorescence intensity, two distinct transition was observed between10-7 to 10-8 and 10-3 to 10-4M and the enzyme activity was disappeared in the presence of Mg2+ below 10-4 M, suggesting that the binding of to the low-affinity site is essential for the catalysis. In literature, the expression of sphingomyelinase of Bacillus cereus was not efficient in E. coli. To improve the yield sphingomyelinase of Bacillus cereus, we have modified the expression and induction system by changing the plating method, lowered temperature, and lowered concentration of IPTG. In this report, we have expressed sphingomyelinase from Bacillus subtilis in E. coli and decreased the amount of sphingomyelinase in inclusion bodies with the yield of 10mg protein in 1L of Bacteria culture.