Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B. thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284----Trp and Met168----Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4 degrees C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolyticaily active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtUis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb Hinfl-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. stearothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8°C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostabiity was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B.caldolyticus.Bacterial species belonging to the genus Bacillus secrete a variety of enzymes, some of which are of economical importance, in particular the ax-amylases and proteinases (5). The various Bacillus species show large variations in optimum growth temperatures, which are often, but not invariably, reflected in the thermostabilities of their extracellular enzymes (23). By comparing thermostable and thermolabile variants of homologous enzymes, information on the mechanisms involved in thermostability of enzymes can be obtained (22,31,37).We selected the neutral proteases from bacilli as a model system in such an approach. Neutral proteases (Npr) are metalloendoproteinases which show optimum activity at neutral pH. These enzymes contain one zinc atom per molecule and may be stabilized by calcium binding (17). Several npr genes have been cloned and expressed in Bacillus subtilis, including the genes from B. subtilis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, and their nucleotide sequences have been determined (8,12,18,33,38,40,44). Recently, the npr gene from Bacillus brevis was also cloned and sequenced (la). Additionally, the primary and tertiary structures of the neutral proteases from Bacillus cereus and Bacillus thermoproteolyticus have been determined (21,28,34,35). The neutral proteases from B. subtilis and B. amyloliquefaciens are rather thermolabile, whereas the enzymes from B. stearothermophilus and ...

Autocatalytic degradation of purified Bacillus subtilis neutral proteinase was examined under various conditions. At elevated temperatures, under non-inhibitory conditions, mature protein was rapidly degraded, but no accumulation of specific breakdown products occurred. However, by incubating purified neutral proteinase on ice during extended periods of time, specific peptides accumulated. These peptides were analysed by SDS/PAGE and Western blotting, and the Nterminal sequences were determined for the four major peptides, which had sizes of 30, 22, 20 and 15 kDa. Sequence data identified five fission sites in the neutral proteinase, three of which were identical with autodigestion target sites in thermolysin, a thermostable neutral proteinase. Comparison of the identified fission sites of the B. subtilis neutral proteinase with the known substrate-specificity of the enzyme indicated that they were in agreement, showing a preference for the generation of fissions at the N-terminal side of large hydrophobic residues, such as leucine, isoleucine and methionine. These results suggest a high degree of similarity in the three-dimensional structures of B. subtilis neutral proteinase and thermolysin.