V.G.H. Eijsink

ENGINEERING A HYPERSTABLE ENZYME BY MANIPULATION OF EARLY STEPS IN THE UNFOLDING PROCESS

Protein engineering experiments have recently yielded hyperstable variants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). These variants contain mutations suggested by comparison of TLP-ste with its more thermostable counterpart thermolysin, as well as rationally designed mutations. The key to the successful stabilization strategy was the identification of a “weak” region that is involved in early unfolding events (“unfolding region”). Mutations in this region had large effects on stability, whereas mutations in other parts of the protein generally had minor effects. The mutational strategies that were used as well as characteristics of the engineered hyperstable biocatalysts are reviewed below.

The effect of site-specific immobilization on the thermal stability of thermolysin-like neutral proteases

Starting from a cysteine-free mutant of the thermolysin-like neutral protease from Bacillus stearothermophilus, cysteines were introduced into different positions on the enzyme surface by site-directed mutagenesis. The mutant enzymes were immobilized via the SH-groups to Activated Thiol-Sepharose 4B and their thermostabilities were compared to those of the soluble enzymes. The results showed that the effects of immobilization on stability strongly depend on the site of attachment. Binding of the enzyme via engineered cysteines in the critical unfolding region between residues 56 and 69 led to a considerable increase of thermal stability, whereas the immobilization via a cysteine introduced remote from the unfolding region yielded less stabilization. An extremely strong stabilization was obtained upon binding via T56C where the half-life at 75 °C was increased by the factor of 24.

STRUCTURAL DETERMINANTS OF THE THERMOSTABILITY OF THERMOLYSIN-LIKE BACILLUS NEUTRAL PROTEASES

Abstract The neutral protease of B. stearothermophilus (NP-ste) is 13 degrees less thermostable than thermolysin, the neutral protease of B. thermoproteolyticus. The sequences of these two proteins differ at 45 positions. By introducing site-directed mutations in NP-ste, it was shown that only a few of these sequence differences account for the total difference in thermostability. All critical residues appeared to be located at the surface of the molecule, mainly in the 63–69 region, and no drastic mutational effects were observed at buried positions. A series of additional mutations was designed that confirmed this trend: mutations in the hydrophobic core of the protein showed only marginal effects, whereas Ala Pro mutations at positions 63, 65, 66 and 69 had pronounced effects on the stability of the enzyme. The results support a model in which it is assumed that local unfolding processes at the surface of the protein, which render the NP susceptible towards autolysis, determine the rate of thermal inactivation. Within the framework of this model mutations that have large effects on stability are expected to be located in regions that unfold relatively easily and that play a role in the early steps of global unfolding. The present results indicate that the 63–69 area in the N-terminal domain of NP-ste is such an early unfolding region.