MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF TWO CORIOLUS VERSICOLOR LACCASE ISOENZYME GENES IN PICHIA PASTORIS

摘要

Laccases have been demonstrated to be potential in several industrial and environmental applications including lignin degradation, biopulping, biobleaching, detoxification of industrial effluent and pollutants, bioremediation, and even organic synthesis. Considering the wide range of applications for laccase, there is a need for clone and screen or mutation laccase genes for novel properties or for large-scale production of selected laccases at low cost. In the present paper, the laccase genes lcc1 and lcc2 encoding for isoenzymes from Coriolus versicolor –nl2304 have been cloned by RT-PCR. These genes display a similarity of 76.39% and 99.80% respectively with other Coriolus versicolor laccases at the amino acid level. The two laccase genes were expressed in Pichia pastoris KM71H with the highest activity of 580U/L (lcc1) and 650U/L (lcc2) when ABTS was used as a substrate. Studies on enzymatic properties showed that the optimum temperatures of the recombinant lcc1 and lcc2 were 50°C, 60°C respectively, and the optimum pH values of the recombinant lcc1 and lcc2 were both 2.0. Both of them were stable at 50°C and at a pH range from 3.0 to 8.0 although the lcc1 was much more stable than lcc2.