By directed evolution and subsequent site-directed mutagenesis, cold-adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four-amino acid substitution variant. RTN29 diplayed a sixfold increase in caseinolytic activity in the temperature range of 15-25 degrees C; a down-shift of optimum temperature by similar to 15 degrees C, as well as a decrease in thermostability; indicating it follows the general principle of tradeoff between acitivity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity,was observed;after heat-treatment at 60 degrees C for 2 h. Notably,. RTN29 exhibited a: lower hydrolytic activity toward suc-AAPF-pNA, due to an increase in K-m and a decrease in k(cat), m contrast to-other artificially cold-adapted subtilases with increased low-temperature activity toward small synthetic substrates. All mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the cold-adapted variants, occurred within or near the substrate-binding g region. None of these mutations; however, match the corresponding sites in-naturally psychrophilic and other artificially, cold-adapted subtilases, implying there are multi, pie routes to cold adaptation. Homology modeling and structural analysis.. demonstrated that these mutations led to an increase in mobility of substrate-binding region and a modulation. of substrate specificity, which seemed to account for the improvement of the enzyme's catalytic activity toward macromolecular substrates at lower temperatures, Our study may provide valuable information needed to develop enzymes coupling high stability and high low-temperature activity, which are highly desired. for industrial use: Biotechnol. Bioeng. 2009;104: 862-870. (C) 2009 Wiley Periodicals, Inc.