Characterization of the refolded laccase from Bacillus sp. HR۰۳

Laccases belong to the multicopper oxidase family. They constitute the most promising enzymes ofoxidoreductase group for industrial applications. In vitro refolding of laccase from inclusion bodies issignificantly important in biotechnology and for structure-function relationship studies. Here, structuraland catalytic features of a refolded laccase from Bacillus sp. HR۰۳ was studied. This laccase wasproduced as a recombinant protein in Escherichia coli (BL-۲۱). Recombinant expression of laccaseleads to the production of protein in both soluble and insoluble form. As laccase is valuable forbiotechnological application, the insoluble form was extracted and refolded. The activity of the refoldedenzyme, using SGZ and ABTS as substrates, was determined. The refolded enzyme showed roughly ۵more times affinity to ABTS than the soluble laccase. Moreover, the catalytic efficiency of refoldedlaccase is about ۷.۴ and ۲.۴ times greater for ABTS and SGZ, respectively. The secondary and tertiarystructure of the enzymes were studied by far-UV circular dichroism spectroscopy and emissionspectroscopy, respectively. Our results also showed a significant difference between the two laccaseforms in terms of thermal stability. Although the refolded laccase was catalytically more efficient, itsthermalstability was not significant compared to that of the soluble laccase.

کلیدواژه ها:

Catalytic efficiency ، Laccase ، Refolding ، Thermal stability