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Additive effects of acyl-binding site mutations on the fatty acid selectivity of Rhizopus delemar lipase

Klein R.R., King G., Moreau R.A., McNeill G.P., Villeneuve P., Haas M.J.. 1997. Journal of the American Oil Chemists' Society, 74 (11) : p. 1401-1407.

DOI: 10.1007/s11746-997-0244-4

The fatty acid specificity and pH dependence of triacylglycerol hydrolysis by the Rhizopus delemar lipase acylbinding site mutant Val206Thr+Phe95Asp (Val, valine; Thr, threonine; Phe, phenylalanine; Asp, aspartic acid) were characterized. The activity of the double mutant prolipase was reduced by as much as 10-fold, compared to the wild-type prolipase. However, the fatty acid specificity profile of the enzyme was markedly sharpened and was dependent on the pH of the substrate emulsion. At neutral pH, strong preference (10-fold or greater) for hydrolysis of triacylglycerols of medium-chainlength fatty acids (C8:0 to C14:0) was displayed by the variant prolipase, with no hydrolysis of triacylglycerols of short-chain fatty acids (C4:0 to C6:0) and little activity manifested toward fatty acids with 16 or more carbons. At acidic pH values, the fatty acid selectivity profile of the double mutant prolipase expanded to include short-chain triacylglycerols (C4:0, C6:0). When assayed against a triacylglycerol mixture of tributyrin, tricaprylin and triolein, the Val206Thr+Phe95Asp prolipase displayed a high selectivity for caprylic acid and released this fatty acid at least 25-fold more efficiently than the others present in the substrate mixture. When presented a mixture of nine fatty acid methyl esters, the wild-type prolipase showed a broad substrate specificity profile, hydrolyzing the various methyl esters to a similar extent. Contrastingly, the double mutant prolipase displayed a narrowed substrate specificity profile, hydrolyzing caprylic methyl ester at nearly wild-type levels, while its activity against the other methyl esters examined was 2.5- to 5-fold lower then that observed for the wild-type enzyme.

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