Title
Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain
Author
TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Levasseur, A.
Pagès, S.
Fierobe, H.P.
Navarro, D.
Punt, P.
Belaïch, J.P.
Asther, M.
Record, E.
Publication year
2004
Abstract
A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergilhis niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from A. niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.
Subject
Biotechnology
Amino acids
Bacteria
Bioassay
Electrophoresis
Enzymes
Fungi
Feruloyl esterase A (FAEA)
Fungal enzymes
Intracellular production
Western blotting
Proteins
Antibody
Bacterial protein
Cell protein
Chimeric protein
Cohesin
Esterase
Fungal enzyme
Glucan 1,4 alpha glucosidase
Hybrid protein
Protein dockerin
Protein feruloyl esterase A
Unclassified drug
Enzyme
Fungus
Microbiology
Protein
Amino acid sequence
Aspergillus niger
Cellular distribution
Clostridium thermocellum
Complex formation
Enzyme activity
Enzyme binding
Enzyme substrate complex
Molecular weight
Nonhuman
Northern blotting
Polyacrylamide gel electrophoresis
Posttranscriptional gene silencing
Protein degradation
Protein domain
Protein processing
Protein protein interaction
Protein secretion
Transcription initiation
Western blotting
Aspergillus niger
Bacterial Proteins
Biotechnology
Carboxylic Ester Hydrolases
Carrier Proteins
Cell Cycle Proteins
Chromosomal Proteins, Non-Histone
Clostridium thermocellum
Fungal Proteins
Nuclear Proteins
Recombinant Fusion Proteins
Transcription, Genetic
Transformation, Genetic
Actinobacteria (class)
Aspergillus
Aspergillus niger
Bacteria (microorganisms)
Clostridium
Clostridium thermocellum
Fungi
Posibacteria
uncultured actinomycete
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http://resolver.tudelft.nl/uuid:e911f0f6-2536-4abc-8f65-17299b1ce063
DOI
https://doi.org/10.1128/aem.70.12.6984-6991.2004
TNO identifier
238234
ISSN
0099-2240
Source
Applied and Environmental Microbiology, 70 (70), 6984-6991
Document type
article