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Journal of Lipid Research, Vol. 41, 846-851, May 2000
Copyright © 2000 by Lipid Research, Inc.

Methods

Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous–organic biphasic system

Correspondence to: Mutsumi Sano

Lysoglycosphingolipids were produced from glycosphingolipids by using sphingolipid ceramide N-deacylase, which cleaves the N-acyl linkage between fatty acids and sphingosine bases in various glycosphingolipids. The enzyme reaction was done in a biphasic media prepared with water;–immiscible organic solvent and aqueous buffer solution containing the enzyme. We investigated the effects of organic solvents and detergents on lysoglycosphingolipid production in the biphasic system. Among the organic solvents tested, n-butylbenzene, cumene, cyclodecane, cyclohexane, n-decane, diisopropylether, n-heptadecane, and methylcyclohexane promoted hydrolysis of GM1, whereas benzene, chloroform, ethyl acetate, and toluene inhibited GM1 hydrolysis. Hydrolysis of asialo GM1, GD1a, GalCer, and sulfatide was also enhanced by the addition of n-decane. The hydrolytic activity of the enzyme was enhanced by the addition of 0.8% sodium taurodeoxycholate or sodium cholate to the aqueous phase. The most effective hydrolysis of various glycosphingolipids by the enzyme was thus obtained in the aqueous-n-decane biphasic system containing 0.8% sodium taurodeoxycholate. Under this condition, the fatty acids released from GM1 by the action of the enzyme were trapped and diffused into the organic phase, while lysoGM1 remained in the aqueous phase.

Thus the almost complete hydrolysis of GM1 was achieved using the biphasic system, while at most 70% of hydrolysis was obtained using normal aqueous media possibly due to the inhibition of hydrolysis reaction by accumulation of fatty acids in the reaction mixture.—Kurita, T., H. Izu, M. Sano, M. Ito, and I. Kato. Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous;–organic biphasic system. J. Lipid Res. 2000. 41: 846;–851.

Supplementary key words: sphingolipid ceramide, N-deacylase, lysosphingolipid, biphasic system, organic solvent, detergent

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