The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents.
Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and detergents. The solvent concentrations where 50% of enzyme activity were irreversibly lost (C(50)) decreased in the order methanol > 2-propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C(50) values were remarkably higher for the thermostable variants than for the wild-type enzymes. Therefore, the stabilization of this loop region also protects the molecule from irreversible inactivation by solvents, and inactivation seems to follow principally the same mechanism as thermal inactivation. However, in contrast to thermal inactivation where the corresponding T(50) values of DM and BLN differed by 10 K, the differences of the C(50) values of DM and BLN were not significant. Detergents had great effects on proteolytic activities which were dependent on the individual detergent and its concentration, but mostly without significant differences between the enzyme variants. These effects were inactivating (SDS, sulfobetaine) or strongly activating (CTAB, CHAPS). Triton X-100 and Tween 20 were activating or inactivating at low and high concentrations, respectively. In all detergents, stabilities of the enzymes were strongly decreased. However, the more thermostable variants were affected by the detergents to the same extent as the wild-type enzymes suggesting that the mechanism of detergent inactivation is different from that of thermal inactivation.
Mansfeld
Johanna
J
Department of Biochemistry/Biotechnology, Martin-Luther University, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany. mansfeld@biochemtech.uni-halle.de
Ulbrich-Hofmann
Renate
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003902:Detergents / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D014644:Genetic Variation; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000737:chemistry*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D012997:Solvents / Q000494:pharmacology; D013696:Temperature; D014867:Water / Q000737:chemistry
D003902:Detergents; D004220:Disulfides; D009930:Organic Chemicals; D012997:Solvents; D014867:Water; D010447:Peptide Hydrolases