Biochemical properties and three-dimensional structures of two extracellular lipolytic enzymes from Bacillus subtilis

This article reviews our present knowledge on the extracellular lipolytic enzymes LipA and LipB from Bacillus subtilis. Growth of B. subtilis to the late logarithmic growth phase results in a total lipolytic activity of 12–18 units per liter of culture supernatant. Immunodetection with LipA- and LipB-specific antibodies indicated a differential expression of both lipolytic enzymes depending on the composition of the growth medium. LipA was produced in rich and in minimal medium, whereas LipB was present only in rich medium. The lipA and lipB genes were cloned and overexpressed in B. subtilis and Escherichia coli, the corresponding proteins purified to electrophoretic homogeneity and their substrate specificities, pH- and temperature stabilities were determined. The active site residue Ser78 of LipB is located in the consensus sequence Ala–X–Ser–X–Gly where the alanine replaces a glycine found in most of the bacterial lipases. The role of this Ala-residue was investigated by constructing LipB variant A76G thereby restoring the canonical lipase consensus motif. When compared with wild-type LipB this variant showed a markedly reduced thermostability at pH 11 but an increased stability at pH 5–7. These findings were rationalized by building a three-dimensional structural model of LipB using the atomic coordinates of the LipA crystal structure, which was solved recently. The LipB model structure revealed that 43 out of 45 residues, which are different from LipA, were located on the surface of LipB. The surface-exposed amino acids including those located at the rim of the active site cleft may cause the differences in specific activities between LipA and LipB.