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 18,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of a recombinant pyranose oxidase (P2O) from the basidiomycete Trametes ochracea has been increased 10-fold in shaking flask cultures of Escherichia coli BL21(DE3) harboring plasmid pSE33 by optimizing the composition of the culture medium using an experimental design approach. Inexpensive lactose was used as a medium component and inducer of expression of the P2O gene, which is under the control of a trc promoter. The expression system was studied in detail in batch and fed-batch cultivations with the aim to improve the expression level of active recombinant protein and to minimize the formation of inclusion bodies. In batch cultivations, the highest specific P2O activity of 0.9 U (mg of soluble protein)(-1) was measured in oxygen-limited cultures grown at 25 degrees C. The highest overall volumetric productivity of 33 mg of active P2O per liter and hour (corresponding to 345U (L h)(-1)) has been determined in a high-density fed-batch process with a feed-forward exponential feeding strategy. During the fed-batch process, lactose was added intermittently to the culture. A final biomass concentration of 33 g L(-1) (based on cell dry weight) was obtained. Compared to shaking flask cultures in not optimized culture media, the overall volumetric P2O productivity has been improved by a factor of 110 using the fed-batch strategy and the optimized culture medium. Recombinant P2O was expressed in the cytoplasm with 9% of the total soluble protein being active P2O. In terms of physical and enzyme kinetic properties, the purified recombinant P2O was found to be similar to the previously published data of P2O isolated from its original host.
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PMID:High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer. 1517 85

Optimization of pyranose-2-oxidase (P2O) production conditions from Trametes versicolor was carried out in shaking cultures containing glucose, malt, and yeast extracts; the optimum concentration values were found to be 1.5% glucose, 1.0% yeast extract, and 1.0% malt extract, pH 5.0, temperature, 26 degrees C, and agitation rate 150 rpm. For the first time, P2O production was also carried out in a stirred tank reactor (STR) with 2.2 L working volume in the optimized medium composition, and biomass, P2O activity, protein, nitrogen and glucose concentrations were also monitored besides pH and dissolved oxygen (DO). In the STR, P2O activity peaked on day 9. Partial enzyme characterization occurred and optimum pH and temperature were detected as 7.0 and 37 degrees C, respectively. K(m) value was found to be 1.009 mM.
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PMID:Pyranose 2-oxidase (P2O): production from trametes versicolor in stirred tank reactor and its partial characterization. 1909 Apr 19

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 degrees 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 x h and 14 U/L x h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L x 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 degrees C with 32 U/L x h. The pCOLD system showed the highest productivity rate (19 U/L x h) at 25 degrees C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25 degrees C with a specific growth rate of mu = 0.15 h-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L x h.
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PMID:Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from Trametes multicolor in E. coli. 2021 72