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Studies on the production of fungal peroxidases in Aspergillus niger

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Author: Conesa, A. · Hondel, C.A.M.J.J. van den · Punt, P.J.
Type:article
Date:2000
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Applied and Environmental Microbiology, 7, 66, 3016-3023
Identifier: 56789
doi: doi:10.1128/AEM.66.7.3016-3023.2000
Keywords: Nutrition · Botany · Fungi · Fungal enzyme · Glucan 1,4 alpha glucosidase · Hemoglobin · Lignin peroxidase · Manganese peroxidase · Messenger RNA · Peroxidase · Recombinant protein · Aspergillus niger · Enzyme activity · Enzyme purification · Enzyme release · Enzyme synthesis · Gene overexpression · Nonhuman · Nucleotide sequence · Protein expression · Protein processing · Aspergillus niger · Blotting, Northern · Blotting, Western · Ferrous Compounds · Heme · Peroxidases · Phanerochaete · Recombinant Proteins · Transformation, Genetic · Aspergillus niger · Chrysosporium · Fungi · Phanerochaete chrysosporium

Abstract

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields. Chemicals/CAS: Ferrous Compounds; Heme, 14875-96-8; lignin peroxidase, EC 1.11.1.-; manganese peroxidase, EC 1.11.1.13; Peroxidases, EC 1.11.1.-; Recombinant Proteins