JBC, Vol. 251, Issue 2, 431-437, Jan, 1976

Hydrolysis of 13-sophorosyloxydocosanoic acid esters by acetyl- and carboxylesterases isolated from Candida bororiensis

M. L. Bucholtz and R. J. Light

Two enzymes have been isolated from Candida bogoriensis which catalyze the hydrolysis of 13-sophorosyloxydocosanoic acid (Glc2HDA) esters obtained from this organism. The 6',6"-diacetyl derivative of Glc2HDA (Ac2Glc2HDA) is hydrolyzed by an acetylesterase (EC 3.1.1.6) which has been purified 1300-fold. The acetylesterase has a molecular weight of 35,000 estimated from gel filtration, and is much more active with p-nitrophenyl acetate than with the acetylated glycolipid. The rate of hydrolysis increases with Ac2Glc2HDA concentration until a plateau is reached at a concentration of about 40 muM, near the critical micelle concentration of this glycolipid. These kinetic data are interpreted as an enzyme specificity for the monomeric, but not the micellar form of the glycolipid. The acetylesterase is inhibited by 0.1 to 10 mM diisopropyl fluorophosphate, 5 mM p-hydroxymercuribenzoate, and 5 mM N-ethylmaleimide, but only slightly by 5 mM iodoacetamide. The methyl ester of Ac2Glc2HDA is hydrolyzed by at least two carboxylesterases (EC 3.1.1) which differ in size according to gel filtration. Their molecular weights are estimated as 140,000 for carboxyesterase A and 40,000 for carboxyesterase B. Both carboxylesterases were purified over 20-fold, and carboxylesterase A was characterized further. Carboxylesterase A activity was inhibited completely by 0.1 to 10 mM diisopropyl fluorophosphate and by 10 mM p-hydroxymercuribenzoate, but only slightly by lower concentrations of p-hydroxymercuribenzoate or by N-ethylmaleimide or iodoacetamide. The carboxylesterase A preparation also acted as a thioesterase with palmityl-CoA (palmityl-CoA hydrolase, EC 3.1.2.2), showing the following approximate relative activities: palmityl-CoA, 100; octanoyl-CoA, 90; methyl Glc2HD, 22; butyryl-CoA, 18; methyl AcGlc2HD, 15; methyl Ac2Glc2HD, 10; and acetyl-CoA, O. Methyl Ac2Glc2HD showed some substrate inhibition at higher concentrations, but neither methyl Ac2Glc2HD nor palmityl-CoA approached enzyme saturation until well above their critical micelle concentrations, indicating hydrolysis of the micellar substrate was occurring. The carboxylesterase and palmityl-CoA hydrolase activities were destroyed in a parallel fashion by heat denaturation, and each substrate inhibited the action of the preparation on the other substrate, but the preparation has not been purified sufficiently to establish with certainty that both activities reside in the same protein.
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