The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

Johanna Mansfeld; Renate Ulbrich‐Hofmann

doi:10.1002/bit.21292

journal article May 24, 2007

The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

Johanna Mansfeld Renate Ulbrich‐Hofmann

Biotechnology and Bioengineering Vol. 97 No. 4 pp. 672-679 · Wiley

View at Publisher Save 10.1002/bit.21292

Abstract

AbstractEngineered 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 (C50) decreased in the order methanol > 2‐propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C50 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 T50 values of DM and BLN differed by 10 K, the differences of the C50 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. Biotechnol. Bioeng. 2007;97: 672–679. © 2006 Wiley Periodicals, Inc.

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Details

Published: May 24, 2007
Vol/Issue: 97(4)
Pages: 672-679
License: View

Authors

J

Johanna Mansfeld

R

Renate Ulbrich‐Hofmann

Cite This Article

Johanna Mansfeld, Renate Ulbrich‐Hofmann (2007). The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents. Biotechnology and Bioengineering, 97(4), 672-679. https://doi.org/10.1002/bit.21292

The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

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