Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H2O2 stability.
One of the major problems of wild-type lignin peroxidase (LiP) is its inactivity at the presence of excess H(2)O(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(2)O(2) was increased over the wild type. The K(m) value of the variants toward H(2)O(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(2)O(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.
Ryu
Kang
K
Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-040, Republic of Korea.
Hwang
Sang Youn
SY
Kim
Ki Hyung
KH
Kang
Jung Hye
JH
Lee
Eun Kyu
EK
D001673:Biodegradation, Environmental; D002733:Chlorophenols / Q000737:chemistry*; D032621:DNA Shuffling / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D024861:Genetic Enhancement / Q000379:methods*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000502:physiology*
D002733:Chlorophenols; D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase; C004762:2,4-dichlorophenol