ADDITIVE EFFECTS OF ACYL-BINDING SITE MUTATIONS ON THE FATTY-ACID SELECTIVITY OF RHIZOPUS-DELEMAR LIPASE

The fatty acid specificity and pH dependence of triacylglycerol hydrol ysis by the Rhizopus delemar lipase acyl-binding site mutant Val206Thr + Phe95Asp (Val, valine; Thr, threonine; Phe, phenylalanine; Asp, asp artic acid) were characterized. The activity of the double mutant prol ipase was reduced by as much as 10-fold, compared to the wild-type pro lipase. However, the fatty acid specificity profile of the enzyme was markedly sharpened and was dependent on the pH of the substrate emulsi on. At neutral pH, strong preference (10-fold or greater) for hydrolys is of triacylglycerols of medium-chainlength fatty acids (C-8:0 to C-1 4:0) was displayed by the variant prolipase, with no hydrolysis of tri acylglycerols of short-chain Fatty acids (C-4:0 to C-6:0) and little a ctivity manifested toward fatty acids with 16 or more carbons. At acid ic pH values, the fatty acid selectivity profile of the double mutant prolipase expanded to include short-chain triacylglycerols (C-4:0' C-6 :0). When assayed against a triacylglycerol mixture of tributyrin, tri caprylin and triolein, the Val206Thr + Phe95Asp prolipase displayed a high selectivity for caprylic acid and released this fatty acid at lea st 25-fold more efficiently than the others present in the substrate m ixture. When presented a mixture of nine fatty acid methyl esters, the wild-type prolipase showed a broad substrate specificity profile, hyd rolyzing the various methyl esters to a similar extent. Contrastingly, the double mutant prolipase displayed a narrowed substrate specificit y profile, hydrolyzing caprylic methyl ester at nearly wild-type level s, while its activity against the other methyl esters examined was 2.5 - to 5-fold lower then that observed for the wild-type enzyme.