Purification and kinetics of a thermostable laccase from Pycnoporus sanguineus (SCC 108)

Pycnoporous sanguineus was identified as a laccase producer when grown on diluted molasses. The single laccase was purified with a purification factor of 967 by ammonium sulphate precipitation, ion exchange and dye affinity chromatography, to a specific activity of 32.9 U/mg. The molecular mass of 58,000Da, the optimum pH range between 3 and 5 with ABTS, DMP and guaiacol as substrates and the isoelectric point of 6.7, was similar to some other laccases of filamentous fungi. The optimum temperature was 55 degrees C and the enzyme displayed enhanced thermal stability with a half-life of 170 min at 75 degrees C. In terms of k(cat)/K-m values for ABTS, syringaldazine and DMP, DMP was the best substrate. Kinetic analysis of fifteen more compounds revealed that, the enzyme was a true laccase with a requirement for a free -OH group with an adjacent free or derivatised -OH. The o-diphenols were preferred above their p-counterparts. Compounds with three adjacent -OH groups displayed higher binding affinities but phloroglucinol, an in-substituted phenol was unreactive. The apparent higher stability of this laccase makes it a good candidate for further investigation into it possible application in biotechnology. (c) 2006 Elsevier Inc. All rights reserved.