Title
Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme
Author
Record, E.
Punt, P.J.
Chamkha, M.
Labat, M.
van den Hondel, C.A.M.J.J.
Asther, M.
Centraal Instituut voor Voedingsonderzoek TNO
Publication year
2002
Abstract
Pycnoporus cinnabarinus laccase lac1 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. Molecular Sequence Numbers: GENBANK: AF170093; Chemicals/CAS: DNA, Complementary; Laccase, EC 1.10.3.2; Oxidoreductases, EC 1.-; Recombinant Proteins
Subject
Nutrition
Amino Acid Sequence
Base Sequence
Basidiomycota
Blotting, Northern
Blotting, Western
Cloning, Molecular
DNA, Complementary
Hydrogen-Ion Concentration
Isoelectric Point
Kinetics
Laccase
Molecular Weight
Oxidoreductases
Recombinant Proteins
Temperature
Aspergillus
Aspergillus niger
Prokaryota
Pycnoporus cinnabarinus
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http://resolver.tudelft.nl/uuid:18ef4465-b049-4cc2-89c3-91923a5e7d99
DOI
https://doi.org/10.1046/j.0014-2956.2001.02690.x
TNO identifier
72516
ISSN
0014-2956
Source
European Journal of Biochemistry, 269 (2), 602-609
Document type
article