Fig. 1. Sequence alignment of lipases and esterases from different Bacillus species. (A) The strictly conserved amino acids of the consensus pentapeptides are boxed. The overall protein sequence similarities of the mature enzymes were determined with the program MegAlign of the software package DNA-Star (Lasergene). Amino acid sequences were retrieved from the GenBank database (http://www.ncbi.nml.nih.gov/) and have the following accession numbers: C69652 (B. subtilis LipB), M74010 (B. subtilis LipA), A34992 (B. pumilus lipase), AJ297356 (B. licheniformis lipase), U35855 (B. licheniformis esterase), AF134840 (B. thermoleovorans lipase), X95309 (B.thermocatenulatus lipase) and U78785 (B.stearothermophilus lipase). (B) Amino acid sequence alignments of four closely related extracellular lipases and esterases from B. subtilis, B. pumilus and B. licheniformis. The residues forming the catalytic triad are marked with an asterisk.

Fig. 2. Extracellular lipolytic activity of B. subtilis 168. (A) Lipase activity was determined with a spectrophotometric assay using p-nitrophenyl-palmitate as the substrate in supernatants obtained from cultures grown in rich medium (Luria broth). The cell density of the cultures grown at 37 °C (▪) and the lipolytic activity in the culture supernatant (♦) were determined over a time period of 45 h. (B) Western-blots with LipA- and LipB-specific antibodies of B. subtilis culture supernatants from cultures grown to the end of the logarithmic growth phase in rich and minimal medium. Ten nanogram each of purified LipA or LipB protein were used as a control.

Fig. 3. 3D structure of LipA and 3D structural model of LipB. (A) Schematic drawing of the structure of B. subtilis lipase LipA. The catalytic triad residues Ser77, His156 and Asp133 are labeled with S, H and D, respectively. The letters N and C indicate the N- and C-termini, respectively. (B) Schematic drawing of the model of LipB. The view is from the solvent into the active site. The catalytic residues Ser78 (S), Asp134 (D) and His157 (H) are indicated in a ball-and-stick representation.

Table 1. Biochemical properties of extracellular lipases and esterases from Bacillus species

Table 2. Specific activities of B. subtilis LipA and LipB towards various substrates

Table 3. Ranking of ratios of specific activities towards various substrates of wild-type B. subtilis esterase LipB and its variant A76G