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

Abstract

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.

Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis

“…The maintenance of protein conformation over a wide concentration range of water-miscible organic solvents has been reported in other enzymes, such as thermolysin [79] and neutral protease [80]. Organic-solvent-tolerant enzymes appear to be quite attractive for industrial applications, such as bioremediation of the organic solvent-contaminated samples.…”

Effects of Site-Directed Mutagenesis of the Loop Residue of the N-Terminal Domain Gly117 of Thermolysin on Its Catalytic Activity

“…FAGLA and ZDFM are substrates routinely used in the characterization of thermolysin 3,14) and neutral proteases from Bacillus stearothermophilus. [37][38][39] FAGLA is a poorly soluble neutral dipeptide, while ZDFM is a negatively charged dipeptide. We have reported several thermolysin variants, L144S, D150E, and I168A, with higher FAGLA-and ZDFM-hydrolyzing activities than WT.…”

Prime Enzymatic Exocellular Background of <i>Lysinibacillus louembei</i>

“…In addition, this stability is interesting between 37˚C -40˚C. Above 50˚C, this enzyme is still stable with a clear decrease in activity despite the fact that the bacteria no longer grow at these temperatures in contrary to studies previously demonstrated [12] [13]. Indeed, the in vitro analysis conditions do not necessarily reflect the physiological conditions in vivo and that environmental factors can positively or negatively influence the protein stability that can explain this result [14].…”