Tailoring CotA Laccase Substrate Specificity by Rationally Reshaping Pocket Edge.

CotA is a bacterial multicopper oxidase, capable of oxidizing lots of substrates. In previous work, small size lignin phenol derivates were found to lay only in the partially covered part of pocket. However, big size substate would occupy the whole pocket to react. In this work, five residues sitting at the edge of the pocket were selected to study their roles in regulating activities against different size substrates. All mutants showed impaired activities against small size sinapic acid, however, A227E, G321F and G321P showed around 25% increase of activities against big size ditaurobilirubin compared to wild type (WT). T262F/G321F showed moderate increased activity to alazin red S. kcat/Kms against ditaurobilirubin of A227E, T262F and G321F are around 1.5, 3 and 1.5 folds of WT's. Unexpectedly, heterologous expression yields of T262F, T262F/G321F and T262F/G321P in Escherichia coli greatly increased by around 5, 7 and 21 folds compared to WT, respectively. It is speculated positive mutants would provide a beneficial orientation for big size substrates. Substituting semi-buried residue T262 by a hydrophobic amino acid might enhance expression yields mainly by increasing van der waals and hydrophobic interaction. This work exemplified rationally regulating specific activities of laccase and is valuable for industrial application.