CopA is a protein formed as part of a copper resistance operon in Pseudomonas syringae pv tomato， but CopA has also been identified from gene library screening as a potential lignin-oxidising enzyme. Few bacterial homologues for bacterial multi-copper laccases have been identified that can assist in lignin degradation. Bioinformatic analysis revealed that copA and copC genes were found in the genomes of bacterial strains capable of lignin oxidation. In this study， CopA enzymes from bacterial strains with lignin oxidation activity， Pseudomonas putida and P. fluorescens， were heterologously expressed and characterised kinetically， and expression of bacterial CopC proteins was also investigated. Purified... More
CopA is a protein formed as part of a copper resistance operon in Pseudomonas syringae pv tomato， but CopA has also been identified from gene library screening as a potential lignin-oxidising enzyme. Few bacterial homologues for bacterial multi-copper laccases have been identified that can assist in lignin degradation. Bioinformatic analysis revealed that copA and copC genes were found in the genomes of bacterial strains capable of lignin oxidation. In this study， CopA enzymes from bacterial strains with lignin oxidation activity， Pseudomonas putida and P. fluorescens， were heterologously expressed and characterised kinetically， and expression of bacterial CopC proteins was also investigated. Purified CopA enzymes were dependent upon exogenous copper (II) ions for activity when expressed under fully aerated conditions， however after expression under microaerobic conditions with copper reconstitution， the activity was independent of copper addition. The CopA enzymes showed activity towards the laccase substrates 2，2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); syringaldazine (SGZ); guaiacol; 2，6-dimethoxyphenol (DMP) and 2，4-dichlorophenol (DCP). Moreover， CopA proteins were able to oxidise the lignin model compounds guaiacylglycerol-beta-guaiacyl (GGE) and 2，2'-dihydroxy-3，3'-dimethoxy-5，5'-dicarboxybiphenyl (DDVA)， giving oxidised dimerised products; and they were active towards Ca-lignosulfonate， giving vanillic acid as product. A double gene deletion of copA-I and copA-II genes in Pseudomonas putida KT2440 was constructed， and this mutant showed diminished growth capability on different small aromatic compounds related with lignin degradation， when copper salts were present in the media.
