Thermostable variants of subtilisin selected by temperature‐gradient gel electrophoresis

Abstract

Region-specific random mutagenesis in the weak calcium binding site of subtilisin Carlsberg and subsequent screening for variants with enhanced heat stability revealed two variants, which showed significantly enhanced residual activity at 68 degrees C, 0.1 mM CaCl2, pH 8.0. Preselected variants have been studied by temperature-gradient gel electrophoresis (TGGE) and were found to be stabilized due to different effects. Whereas the point mutation (Ser188Pro) mainly enhanced autoproteolytic stability of subtilisin, the double mutation (Ser188Pro; Ala194Glu) additionally increased the apparent Tm-value of the molecule for 2-3 degrees C under a variety of conditions. It was possible to differentiate between the effects of autoproteolysis and structural unfolding to a certain degree by TGGE and to show the complex influence of changed calcium affinity on thermal stability for the double variant.

Molecular markers of serine protease evolution

“…The numerical preference for Pro225, which prevents cation binding, may be an example of a conserved proline that regulates the folding kinetics of secondary structural elements (Hardy and Nelson, 2000). In subtilisin-type proteases, mutation of residue 188 to Pro, just upstream of the conserved marker Ser190, results in enhanced thermostability (Sa Èttler et al, 1996) and the ability to function in denaturing environments (Chen and Arnold, 1993). Ser190 is located near the active site and substrate binding pocket, implying that the loop containing the Ser190 marker is associated with proper folding of the catalytic apparatus.…”

Proteases in Organic Synthesis

“…As a result, purely random evolution of a biocatalyst is especially useful to improve more global (cumulative) properties, such as stability, rather than the native activity or specificity of an enzyme, which usually depends on synergistic mutational events. Recent studies on the improvement of protease stability came, for example, from Sattler et al and added to a list of earlier studies in this field . The authors randomly mutated subtilisin and successfully screened for enhanced thermostable variants by temperature-gradient gel electrophoresis.…”

“…Recent studies on the improvement of protease stability came, for example, from Sattler et al 275 and added to a list of earlier studies in this field. 276 The authors randomly mutated subtilisin and successfully screened for enhanced thermostable variants by temperature-gradient gel electrophoresis.…”

Thermal stable and oxidation-resistant variant of subtilisin E

“…Unfortunately, only a few mutants with high thermostability have been found at present due to the limited knowledge of the structure-stability relationship of the investigated protein, although thermostability is correlated with the increased number of residues involved in charge-charge and aromatic -aromatic interactions. The random mutagenesis in vitro is an efficient approach when coupled with an efficient screening of selection procedures to identify colonies expressing variant enzymes with the properties of interest (Strausberg et al, 1995;Sattler et al, 1996;Takagi et al, 1996Takagi et al, , 1997Takagi et al, , 1998You and Arnold, 1996). One thermally stable variant of subtilisin BPN%, N218S was identified using a simple plate assay procedure to screen for esterase activity on nitrocellulose filters after preincubation at elevated temperature (Bryan et al, 1986).…”