Patterns of adaptation in a laboratory evolved thermophilic enzyme

Abstract

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°C 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 8-4A9 at 60°C 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 8-4A9 unfolds at temperatures approx. 25°C higher than wild-type. It is also substantially more resistant to proteolysis at 30°C. Nearly half of the 13 amino acid substitutions accumulated in 8-4A9 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.

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