To ascertain whether position 131 of a mesophilic protease, subtilisin BPN', is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492-495, 1998), a full set of subtilisin BPN' mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10°C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN' and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (k_cat/K_m) at 10°C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in k_cat and a decrease in K_m. All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10°C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60°C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.