The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k(cat) /K(m)) by 290% at 0 degrees C. In the presence of 1 M TMAO, the thermostability (t(1/2)) of MCP-01 increased by two- to fivefold at 60 approximately 40 degrees C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40 degrees C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T(m)) of MCP-01 by 10.5 degrees C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.