tailieunhanh - Báo cáo khoa học: The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments

It is scientifically and industrially important 1 to clarify the stabilizing mechanism of proteases in extraordinary envi-ronments. We used subtilisins ALPI and Sendai as models to study the mechanism. Subtilisin ALPI is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilicBacillus, whereas sub-tilisin Sendai from alkalophilic Bacillusis stable under conditions of high alkalinity. | Eur. J. Biochem. 269 4577-4585 2002 FEBS 2002 doi The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments Youhei Yamagata1 Hiroshi Maeda1 Tasuku Nakajima1 and Eiji Ichishima2 1 Laboratory of Molecular Enzymology Division of Life Science Graduate School of Agricultural Science Tohoku University Aoba-ku Sendai Japan 2Department of Biotechnology Faculty of Engineering Soka University Hachioji Tokyo Japan It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is exli emlly sensitive to highly alkaline conditions even though the enzyme is produced by alkalophilic Bacillus whereas sub-tilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALPI enzymes by mutating the amino acid residues specific for subtilisin ALPI to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C-terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and on the basis of three-dimensional modelling that they are involved in stabilizing the conformation of the C-terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes. Keywords alkalophilic alkaline resistance Bacillus molecular surface .