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A subtilisin excreted by the Antarctic Bacillus TA39 has been purified to homogeneity and characterised. Two independent genes subt1 and subt2 are present but only subt1 is expressed significantly in the culture medium. The enzyme displays the usual characteristics of cold enzymes i.e. a high catalytic efficiency at low and moderate temperatures and an increased thermosensitivity originating from a 3D structure probably more flexible than its mesophilic counterpart. This is corroborated by the analysis of the computerized structure which shows a significant decrease in the number and strength of intramolecular weak bonds such as salt bridges and aromatic interactions. The affinity for calcium is also almost three orders of magnitude lower than that of mesophilic subtilisin and the interactions with the solvent are significantly higher thanks to a large increase in the number of Asp residues in the loops connecting secondary structures. The relation between flexibility and activity was investigated by site-directed mutagenesis tending mainly to increase the rigidity of the molecular edifice through the incorporation of additional salt bridge, disulfide bridge, aromatic interaction and by increasing the affinity of the enzyme for calcium. An important stabilization of the molecular structure was achieved through a modification of a calcium ligand T85D. The thermostability of the mutated product expressed in a mesophilic Bacillus reaches that of mesophilic subtilisin. Most important is the fact that this mutation further enhances the specific activity by a factor close to 2 when compared to the wild type enzyme so that the overall activity of the mutated cold enzyme is about 20 times higher than that of mesophilic subtilisin, illustrating the fact that thermostability is not systematically inversely related to specific activity. This opens new perspectives in the use of cold enzymes in biotechnology.

protein engineering, design and selection

Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold