ObjectiveRational engineering of the crevice-like binding site of lipases for improvement of lipases' catalytic properties.ResutsThe residues located at the crevice-like binding site of four representative lipases including Thermomyces lanuginosus lipases (TLL and Lip), Rhizopus oryzae lipase (ROL), and Rhizomucor miehei lipase (RML) were identified through structural analysis. The residues at the bottom of the crevice-like binding site recognizing the substrates with short/medium carbon chain length and those located at the right-hand wall of the surface crevice region affecting the product release were changed by site-directed mutagenesis. The corresponding double mutants exhibited 5 to 14-fold higher activity towards p-nitrophenyl esters than their wild types, and their substrate preference shifted to acyl moiety with shorter carbon chain length. In addition, the mutations led to an increase of B-factor, resulting in decrease of their optimum temperature by 10-20 degrees C.ConclusionsThe key residues located at the crevice-like binding site play important roles in determining lipase activity, substrate preference and optimum temperature, which offers a useful new paradigm for facilitating rational design of lipases.