Patterns of adaptation in a laboratory evolved thermophilic enzyme

The heat sensitive psychrophilic protease subtilisin S41 was previously subjected to three rounds of mutagenesis/ recombination and screening, resulting in variant 3-2G7, whose half-life at 60 degreesC is approx. 500 times that of wild-type. Here we report the results of five additional generations of laboratory evolution starting from 3-2G7. The half-life of 8th generation enzyme 84A9 at 60 degreesC is 1200 times that of wild-type, and slightly more than twice that of 3-2G7. This half-life is > 20-fold greater than those of homologous mesophilic subtilisins SSII and BPN '. Circular dichroism melting curves indicate that subtilisin 84A9 unfolds at temperatures approx. 25 degreesC higher than wild-type. It is also substantially more resistant to proteolysis at 30 degreesC. Nearly half of the 13 amino acid substitutions accumulated in 84A9 involve the mutation of serine residues. This mirrors a pattern observed in natural proteins, where serines are statistically less prevalent in thermophilic enzymes compared to mesophilic ones. (C) 2001 Elsevier Science B.V. All rights reserved.