Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase inthe aqueous-organic biphasic system

Lysoglycosphingolipids were produced from glycosphingolipids by using sphin golipid ceramide N-deacy-lase, which cleaves the N-acyl linkage between fat ty acids and sphingosine bases in various glycosphinolipids. The enzyme rea ction was done in a biphasic media prepared with water-immiscible organic s olvent and aqueous buffer solution containing the enzyme. We investigated t he effects of organic solvents and detergents on lysoglycosphingolipid prod uction in the biphasic system. Among the organic solvents tested, n-butylbe nzene, cumene, cyclodecane, cyclohexane, n-decane, diisopropylether, n-hept adecane, and methylcyclohexane promoted hydrolysis of GM1, whereas benzene, chloroform, ethyl acetate, and toluene inhibited GM1 hydrolysis, Hydrolysi s of asialo GM1, GD1a, GalCer, and sulfatide was also enhanced by the addit ion 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 e nzyme was thus obtained in the aqueous-n-decane biphasic system con taining 0.8% sodium taurodeoxycholate. Under this condition, the fatty acids relea sed 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 almos t 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. Kato. Enhancement of hydrolytic activity of sphin golipid ceramide N-deacylase in the aqueous-organic biphasic system.