Bertus van den Burg

Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond

The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

Selection of mutations for increased protein stability

There are many ways to select mutations that increase the stability of proteins, including rational design, functional screening of randomly generated mutant libraries, and comparison of naturally occurring homologous proteins. The protein engineers toolbox is expanding and the number of successful examples of engineered protein stability is increasing. Still, the selection of thermostable mutations is not a standard process. Selection is complicated by lack of knowledge of the process that leads to thermal inactivation and by the fact that proteins employ a large variety of structural tricks to achieve stability.

One-step affinity purification of Bacillus neutral proteases using bacitracin-silica.

A purification procedure for neutral proteases from bacilli is described, in which bacitracin-silica was used as affinity medium. This enabled a one-step purification of the proteases directly from culture supernatant. Since neutral proteases are extremely sensitive towards autodigestion, conditions were chosen such, that autodigestion was largely prevented. Isopropanol appeared to be useful in both eluting the enzymes from the affinity medium, and inhibiting enzymatic activity during this step. The bacitracin-silica medium allowed high flow rates: with columns prepared for use in an FPLC system flow rates up to one column volume per minute were feasible, and still gave satisfactory results. The neutral proteases purified by this method were found to be homogeneous both by SDS-PAGE and analytical gel filtration.

Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

The members of the M4 peptidase family are involved in processes as diverse as pathogenicity and industrial applications. For the first time a number of M4 family members, also known as thermolysin-like proteases, has been characterized with an identical substrate set and a uniform set of assay conditions. Characterization with peptide substrates as well as high performance liquid chromatography analysis of β-casein digests shows that the M4 family is a homogeneous family in terms of catalysis, even though there is a significant degree of amino acid sequence variation. The results of this study show that differences in substrate specificity within the M4 family do not correlate with overall sequence differences but depend on a small number of identifiable amino acids. Indeed, molecular modeling followed by site-directed mutagenesis of one of the substrate binding pocket residues of the thermolysin-like proteases ofBacillus stearothermophilus converted the catalytic characteristics of this variant into that of thermolysin.

Engineering thermolysin-like proteases whose stability is largely independent of calcium

© 1997 Federation of European Biochemical Societies.

Improving the thermostability of the neutral protease of Bacillus stearothermophilus by replacing a buried asparagine by leucine

Amino acids buried in the hydrophobic interior of a protein with polar side chain atoms, which are not involved in hydrogen bonding or electrostatic interactions, have an adverse effect on protein stability. Replacing such residues by hydrophobic ones may render a protein more stable. Asparagine 241, which is buried in the neutral protease of Bacillus stearothermophilus, was replaced by leucine by side‐directed mutagenesis. This mutation increased the stability of the protein by 0.7 ± 0.1 degree.