Abstract
The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.
Highlights
Enzymatic catalysis provides tremendous opportunities for industry to carry out efficient and economical biocatalytic conversions
Apart from the T7 RNA polymerase based pET21d+ [24], we investigated the L-arabinose inducible pBAD [25,26] and the pCOLD expression system [27,28]
The pET21d+ and the pCOLD system were induced by addition of 0.5% lactose (w/v); for the pBAD system, an L-arabinose concentration of 1 g/L (0.1% w/v, Fig. 1) resulted in the highest expression of active pyranose 2-oxidase (POx)
Summary
Enzymatic catalysis provides tremendous opportunities for industry to carry out efficient and economical biocatalytic conversions. Traditional markets include the food and feed industry, but enzymatic processes are increasingly implemented in emerging markets such as fine chemical production and pharmaceutical industries [1]. Cost-efficient high-yield production of the biocatalysts, usually by heterologous expression in bacterial or yeast systems, is critical for the economic viability of such processes. If posttranslational modifications are not required, prokaryotic systems like E. coli are most attractive due to high productivity, low cost and easy handling. Overexpression of recombinant proteins in bacteria can cause problems like the production of insoluble aggregates of misfolded and inactive proteins called inclusion bodies (IB). Inclusion bodies consist of the overproduced polypeptide aggregated with small amounts of foreign
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