A new way of enhancing the thermostability of proteases.
Thermostability of enzyme can be enhanced by single amino acid substitutions. Recent advances in genetic engineering have made it possible to create novel proteins in a predictable manner where structural information for the protein is available. This 'protein engineering' has already been used to enhance enzyme thermostability, but it is usually not clear which amino acid substitutions should be made. We consider that the following approach should be helpful in engineering proteins with enhanced thermostability: highly conserved residues should be left unchanged; the sequences of known mesophilic and thermophilic proteins should be used to suggest the kinds of changes likely to increase thermostability; and substitutions should be made that increase internal hydrophobicity and that stabilize helices for strong internal packing. We describe here the use of this approach to alter the thermostability of the thermostable neutral protease of Bacillus stearothermophilus, the sequence of which is known. Surprisingly we find that a single mutation that decreases thermostability can require two mutations that increase stability to compensate for it. The effects on stability are not additive, suggesting cooperativity.
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D004795:Enzyme Stability; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013820:Thermolysin / Q000378:metabolism
D010447:Peptide Hydrolases; D013820:Thermolysin