CotA Laccase Production From Recombinant Escherichia Coli For Efficient Bio-catalysis

Laccases are important members of the multi-copper oxidases family.As environmentally friendly biocatalysts,laccases can degrade a broad range of industrial toxic contaminants.Recently,bacterial laccases have drawn researchers' interest due to their catalytic characteristics under high pH and temperature conditions.The major challenges for heterologous expression of bacterial laccases,especially CotA laccase,are the high cost and low production yield.Therefore,development of efficient technologies which are cost effective is necessary for enhancing CotA laccase production for the industrial applications.CotA laccase gene with/without TTA codon was ligated in pET30a and PT7-FLAG-MAT-TAG-1 expression vectors.The recombinant vectors were transformed into Escherichia coli.Among all transformed cells,the recombinant E.coli cells carrying pT7-FLAG-MAT-TAG-1-CotA without TTA codon insertion showed the highest volumetric activity of 1474 U/L.SDS-PAGE results confirmed efficient CotA laccase expression in the recombinant cells.An efficient micro-aerobic cultivation strategy for enhancing CotA laccase expression was developed.Micro-aerobic cultivation of E.coli BL21(DE3)with pT7-FLAG-MAT-TAG-1-CotA significantly enhanced CotA laccase production up to 13903 U/L.The micro-aerobic cultivation strategy enhanced the intracellular reactive oxygen species(ROS)level that consequently led to the over-expression of CotA laccase gene.The resulted CotA laccase showed efficient decolorization performance on malachite green,crystal violet,congo red and bromophenol blue at pH 6 and 9.Functionalized magnetic graphene oxide(MGO)support attached with Na,Na-Bis(carboxymethyl)-L-lysine hydrate(NTA-NH2)and chelated with Cu2+ was synthesized.The Cu2+-chelated MGO(MGO-NTA-Cu2+)exhibited the highest adsorption capacity of 177 mg/g-support among all synthesized nano-composites.The maximum activity recovery of laccase using MGO-NTA-Cu2+ was 114%.The catalytic properties of MGO-NTA-Cu-CotA laccase were significantly improved in comparison with those of free laccase.MGO-NTA-Cu-CotA laccase showed efficient decolorization rate for Congo red(CR)reached 100%after 5 h reaction at 60? and pH 8.MGO-NTA-Cu-CotA laccase retained 89.4%of its initial activity after 10 consecutive cycles.These results prove that MGO-NTA-Cu2+ nano-composite is a promising support for efficient CotA laccase immobilization.In conclusion,the above results provide efficient strategies to enhance CotA laccase expression using strong promoter,efficient expression vector,and novel micro-aerobic cultivation method.Furthermore,nanotechnology can significantly enhance CotA laccase activity for expanding its application in practice.