Abstract
One of the major problems of wild-type lignin peroxidase (LiP) is its inactivity at the presence of excess H 2O 2 and high concentration of aromatic compounds. Little is known about the substrate-binding site of LiP, and functionality improvement of LiP was not actively tried by genetic engineering and directed evolution. In order to improve LiPs functionality, we performed directed evolution with a colorimetric screening method. Finally, three types of LiP mutants were screened. The catalytic efficiency of the variants toward 2,4-dichlorophenol (DCP) degradation activity and the stability against H 2O 2 was increased over the wild type. The K m value of the variants toward H 2O 2 was increased, but K m value toward 2,4-DCP degradation was reduced. Overall, The K cat/ K m values of the mutants toward 2,4-DCP was increased ca. 4-fold, and that toward H 2O 2 was increased ca. 89-fold. Amino acid sequence analysis indicated that the most of the mutations were located on the enzyme surface. We expect that these results coupled with recombining mutation can be successfully applied to the molecular evolution cycles for screening of LiPs and other oxidative enzymes with improved functionality and stability.
Published Version
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