Directed evolution study of temperature adaptation in a psychrophilic enzyme

Abstract

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 shuf¯ing) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacri®cing lowtemperature 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 ef®ciency, 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 re¯ects the effects of evolution, rather than any intrinsic physicalchemical limitations on proteins