Cloning and Expression of Four Laccase genes isolated from Arctic marine Psychrobacter strains in Escherichia coli and psychrophilic Pseudomonas
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A group of diverse bacterial isolates were discovered in Arctic marine habitat during a research expedition in May 2009. These isolates were identified and sequenced based on 16S rRNA sequencing. Psychrobacter strains discovered among the isolates were investigated further for their laccase-like multicopper oxidase activity. Four of the strains P11f6, P11G3, P11G5 and P2G3 were found to show laccase activity. The genes responsible for the laccase-like multicopper oxidase activity were identified and sequenced. The laccase genes were designated P11f6, P11G3, P11G5, P2G3 and their genomic DNA was isolated. The discovery of these Psychrobacter strains and subsequent laccase activity shown by the strains, form the basis of this project.In this project, the four laccase genes, P11f6, P11G3, P11G5 and P2G3 were cloned and expressed in two expression hosts, E. coli BL21 (DE3) and psychrophilic Pseudomonas Psm26D7. The expression hosts harbouring the cloned gene were induced for protein expression and cultivated at varying temperatures and incubation periods. Protein expression levels in E. coli BL21 (DE3) carrying the laccase genes were analysed by SDS-PAGE, results showed that P11f6 and P11G5 laccase genes were expressed. While, P11G3 and P2G3 were not expressed in E. coli BL21 (DE3).The four laccase genes were then introduced into psychrophilic Pseudomonas Psm26D7 by conjugation, followed by induction for protein expression. SDS-PAGE analysis of protein expression levels indicates P11G3, P11G5, and P2G3 laccase genes were expressed in psychrophilic Pseudomonas Psm26D7. Expression of P11f6 in Pseudomonas Psm26D7 was not successful.