We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60°C is ∼500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by ∼10°C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efficiency, k_(cat)/K_M, was enhanced ∼threefold over a wide temperature range (10 to 60°C). The activation energy for catalysis, determined by the temperature dependence of k_(cat)/K_M in the range 15 to 35°C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins.

© 2000 Academic Press. Received 25 October 1999, Revised 4 February 2000, Accepted 18 February 2000. We thank Yi Tang for assistance with CD experiments. We also thank the Caltech Biopolymer Synthesis and Analysis Resource Center for DNA synthesis and the Caltech DNA Sequencing Core Facility for DNA sequencing. K.M. acknowledges the National Institute of Bioscience and Human Technology (Japan) for its generous financial support. This research was supported by Procter & Gamble.