High-alkaline proteases are of great importance because of their proteolytic activity and stability under high-alkaline condition. We have previously isolated a new protease (AprB) which has potential industrial applications based on its high-alkaline adaptation. However, the molecular and structural basis for alkaline adaptation of this enzyme has not been fully elucidated. In the present study, AprB gene was cloned and expressed in the Bacillus subtilis WB600. This gene codes for a protein of 375 amino acids comprised with a 28-residual signal peptide, a 78-residual pro-peptide, and a 269-residual mature protein. The deduced amino acid sequence has the highest homology of 63.2% with that of the high-alkaline proteases. Recombinant AprB was purified and determined to be monomeric with molecular mass of 26.755kDa. The NH(2)-terminal sequence of the purified AprB was A-Q-S-I-P-W-G-I-E-R. This enzyme exhibited high catalytic efficiencies (K(cat)/K(m)) towards natural, modified, and synthesis substrates with optimal activity at 60°C and pH 10. AprB was stable over a wide range of pH 5 to 11 and various surfactants, and could be activated by Mg(2+), Ca(2+) and Ba(2+). The structural properties of AprB, like a higher ratio of R/(R+K), a larger area of hydrophobic surface, increased number of ion pairs formed by Arg residue, and the exposure of Asp active residue on the surface, might be responsible for its alkaline adaptation. In contrast with members of subtilisin family, such as M-protease and subtilisin BPN', AprB harbored a high content of Glu and Asp residues, and a low content of Arg and Lys residues on the surface. Interestingly, these structural characters were similar with that of psychrophilic proteases, which suggested that these molecular factors were not restricted in the psychrophilic proteases, and therefore were not solely responsible for their cold-adaptation. Our results reveal a novel structural feature of AprB unique to subtilisin family and provide clues for its alkaline adaptation.