Abstract
Copper though an essential element, poses serious consequences at elevated concentrations. The bacterial cell utilizes a battery of copper detoxification and exclusion mechanisms of which multicopper oxidase (CueO) is an integral component. In addition to acting as copper regulatory elements, CueOs have been shown to possess laccase activity. In this study, we have cloned and over-expressed the CueO of locally isolated Klebsiella pneumoniae KW strain. The CueO protein was purified to homogeneity by nickel affinity chromatography. Enzyme assays of CueO protein with phenolic substrates revealed its laccase activity. The kinetic studies showed Km value of 0.2 mu M, kKcat 0.68 S-1 and Kcat/km 1.2S(-1) mu M-1 for 2,6-Dimethoxyphenol (DMP) and Km value of 0.25mM, Kcat 300 S-1 and Kcat/Km=1200S(-1)mM(-1) for Syringaldazine (SGZ). Regulation of cueO in response to various concentrations of copper was studied at transcriptional level. Quantitative analysis through real time PCR demonstrated that the mRNA level increased enormously - up to 18.3 times - under copper induction. Time course study revealed a bimodal pattern of expression with two maxima, first at 15 min and second at 90 min exposure time. The role of CueO in copper detoxification as well as its laccase activity makes it suitable for biotechnological applications.