Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from <it>Trametes multicolor </it>in <it>E. coli</it>
2010
Ludwig Roland | Posch Gerald | Spadiut Oliver | Haltrich Dietmar | Peterbauer Clemens K
<p>Abstract</p> <p>The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host <it>E. coli </it>was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d<sup>+</sup>, 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<sup>+ </sup>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<sup>+ </sup>and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d<sup>+ </sup>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<sup>-1</sup>resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.</p>
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