tailieunhanh - Báo cáo khoa học: Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme

Pycnoporus cinnabarinuslaccaselac1genewasoverexpressed in Aspergillus niger, a well-known fungal host producing a large amount of homologous or heterologous enzymes for industrial applications. The corresponding cDNA was placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter as a strong and constitutive pro-moter. The laccase signal peptide or the glucoamylase preprosequence ofA. | Eur. J. Biochem. 269 602-609 2002 FEBS 2002 Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme Eric Record1 Peter J. Punt2 Mohamed Chamkha3 Marc Labat3 Cees A. M. J. J. van den Hondel2 and Marcel Asther1 1Unite INRA de Biotechnologie des Champignons Filamenteux IFR-IBAIM Universites de Provence et de la Méditerranée ESIL Marseille France department of Applied Microbiology and Gene Technology TNO Nutrition and Food Research Institute Zeist the Netherlands 3Unite IRD de Biotechnologie Microbienne Post-Recolte IFR-IBAIM Universites de Provence et de la Mediterranee ESIL Marseille France Pycnoporus cinnabarinus laccase lacl gene was overexpressed in Aspergillus niger a well-known fungal host producing a large amount of homologous or heterologous enzymes for industrial applications. The corresponding cDNA was placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter as a strong and constitutive promoter. The laccase signal peptide or the glucoamylase preprosequence of A. niger was used to target the secretion. Both signal peptides directed the secretion of laccase into the culture medium as an active protein but the A. niger preprosequence allowed an 80-fold increase in laccase production. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis and N-terminal sequencing. The molecular mass of the mature laccase was 70 kDa as expected similar to that of the native form suggesting no hyperglycosylation. The recombinant laccase was purified in a three-step procedure including a fractionated precipitation using ammonium sulfate and a concentration by ultrafiltration followed by a Mono Q column. All the characteristics of the recombinant laccase are in agreement with those of the native laccase. This is the first report of the production of a white-rot laccase in A. niger. Keywords laccase Pycnoporus cinnabarinus heterologous .