| Summary: | B. gibsonii Alkaline Protease (BgAP) is a recently described subtilisin with activity and stability properties suitable for applications in washing detergents, having a significant decrease of activity at low temperatures. To improve BgAP activity at 15°C, directed evolution was used using SeSaM mutagenesis method. A parallel screening towards improved protein stability and further recombination yielded variant 39IC1+N253D with a half time at 60°C more than 200 times that of WT BgAP. Recombination of both sets of amino acid substitutions resulted in variant 39IC1 MutIII with 1.5 times the specific activity and 100 times the half life at 60°C of the WT BgAP. Analysis of the introduced amino acid substitutions revealed no substitutions close to the active site, and activity related ones were non-charged to non-charged of the large to small type whereas those related with improved thermal stability introduced negative charged residues in loop areas, and increasing ionic and hydrogen bonds interactions. To further study protein adaptation, the psychrophilic subtilisin S41 from the Antarctic bacillus TA41, and two variants with two and seven amino acid substitutions were studied using Molecular Dynamics simulation at 283K and 363K. Essential dynamics analysis evidenced that the most important collective motions, especially at 363K, differ in distribution and intensity for each protein. At high temperature and for the thermo labile wild type, selective amplification of a subset of the low temperature largest collective motions was observed. The thermostable variants showed a different pattern of the collective motions at 363K from those at 283K. These results support the hypothesis that these amino acid substitutions, rather than improving the global stability of the variants by increasing its rigidity, selectively activate collective fluxional modes. A better understanding of this process can open alternative strategies to design proteins with improved thermal stability.
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