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

Robert R. Klein, Gregory King, Robert A. Moreau, Gerald P. McNeill, Pierre Villeneuve, Michael J. Haas

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16 Scopus citations


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 25fold 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.

Original languageEnglish
Pages (from-to)1401-1407
Number of pages7
JournalJAOCS, Journal of the American Oil Chemists' Society
Issue number11
StatePublished - 1997
Externally publishedYes


  • Fatty acid specificity
  • Lipase
  • Methyl ester hydrolysis
  • Molecular modeling
  • Mutagenesis
  • Protein structure
  • Triacylglycerol hydrolysis


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