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Gewählte Publikation:

Sygmund, C; Gutmann, A; Krondorfer, I; Kujawa, M; Glieder, A; Pscheidt, B; Haltrich, D; Peterbauer, C; Kittl, R; .
(2012): Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris.
Appl Microbiol Biotechnol. 2012; 94(3):695-704 FullText FullText_BOKU

Abstract:
Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L-1 PDH or 1,330 U L-1 d(-1) in space-time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.
Autor*innen der BOKU Wien:
Haltrich Dietmar
Kittl Roman
Krondorfer Iris
Peterbauer Clemens Karl
Sygmund Christoph
BOKU Gendermonitor:

Find related publications in this database (using NML MeSH Indexing)
Agaricus/enzymology*;Agaricus/genetics;Carbohydrate Metabolism*;Gene Expression*;Oxidoreductases/biosynthesis*;Oxidoreductases/genetics;Pichia/genetics*;Recombinant Proteins/biosynthesis;Recombinant Proteins/genetics;

Find related publications in this database (Keywords)
Pyranose dehydrogenase
Pichia pastoris
Heterologous expression
Agaricus
Large scale
Microscale


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