Lipases and esterases are important biocatalysts for synthetic organic fine chemistry. An esterase from Bacillus sp. BP-7 (EstBP7) bears in its amino acid sequence a rare GGG(A)X oxyanion hole motif, where an uncommon threonine (T) is found at the third position. Detection of this pattern motivated evaluation of the ability of EstBP7 for conversion of tertiary alcohols. The enzyme was engineered in order to optimize its performance to provide important chiral building blocks: five variants with mutations in the oxyanion hole motif were created to investigate the influence on activity and enantioselectivity in the kinetic resolution of eight acetates of tertiary alcohols. Wild-type enzyme converted all esters of tertiary alcohols assayed with low enantioselectivity, whereas some of the mutants displayed significantly increased E-values. One of the mutants (EstBP7-AGA; Mut 5) showed an E > 100 towards a complex tertiary alcohol acetate (2-(4-pyridyl)but-3-yn-2-yl acetate) at low reaction temperature (4 A degrees C). Therefore, the catalytic toolbox was expanded for biocatalysis of optically pure tertiary alcohols valuable for the pharmaceutical industry.