BioSilta Oy FI-90014 Oulu, Finland T: +358-8-5532361 F: +358-207-001500 E: info@biosilta.com
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BioSilta presented a poster at the 25th Annual The Protein Society Meeting in Boston.
The authors of this novel research using EnPresso tablets in the cultivation of Bacillus subtilis showed improved expression and activity of β-galactosidase posterare
Conclusions:
Dr. Antti Vasala presented a poster at the 10th Yeast Lipid Conference in Oulu, Finland.
Pichia pastoris can yield high biomass and product yields in bioreactor cultivations, which are used with chemically defined media and optimized feeding strategies. In shake flasks cultures, however, cultivations often fail to reach high product yields consistently, because the growth rate, pH levels, nutrient feeding or inducer feeding cannot be controlled. Here, we present a novel tablet medium based on patented EnBase technology (enzymatic glucose delivery), which mimics the fed-batch technology generally used in bioreactors. We show that with this new medium composition, significant improvements in cell and product yields can be achieved.
• Slow enzymatic glucose delivery significantly improves cell and product yields. BioSilta has optimized the medium composition to provide stable pH and further improved product yields.
• EnBase is compatible with expression by AOX1-promoter (methanol induction) and GAP-promoter
• The optimized medium composition provides a robust and adjustable environment for recombinant protein production
The EnPresso Tablet uses the EnBase technology, which is based on enzymatic release of glucose from a soluble polymer. The growth rate is controlled by the amount of glucose and can be adjusted by the concentration of the glucose-releasing enzyme (EnZ I’m). This EnBase technology can provide fed-batch-like growth conditions in small scale shaken cultures. The sterilized EnPresso tablets dissolve fast in water to constitute ready-to-use high cell density medium.
THe findings from this poster include:
The optimal glucose feeding rate is dependent on the efficiency of aeration. In EnPresso cultures glucose release rate can be easily adjusted by the concentration of the glucose releasing enzyme to be lower in Erlenmeyer flasks and higher in the highly aerated Ultra Yield Flasks.
Kaisa Ukkonen presented this poster at the 6TH Conference on Recombinant Protein Production in Vienna, Austria
Shake flask cultivations often fail to produce enough correctly folded and biologically active recombinant protein because of uncontrolled growth, non-optimal pH, oxygen depletion and low cell yield. These problems can be tackled with EnBase® technology, which applies fed-batch principle by controlled enzymatic glucose release in shaken cultures and has been shown to significantly enhance the yield of several recombinant proteins expressed in Escherichia coli. Here it is demonstrated that coupling of the EnBase technology with improved oxygen transfer can lead to further significantly enhanced results.
Medium based on enzymatic glucose control is superior to conventional media such as Terrific Broth or ZYM autoinduction: volumetric yield of active recombinant ADH was 60 to 140 fold increased in EnPresso medium in ordinary Erlenmeyer flasks.
Even further yield improvement can be achieved by combining EnPresso with enhanced oxygen transfer. EnPresso + Ultra Yield Flask + Enhanced Seal combination provided 100 to 200 fold higher yield of active ADH in comparison to TB or ZYM.
Improved oxygen transfer in TB or ZYM by Ultra Yield Flask or by the novel Enhanced Seal in Erlenmeyer flask provided cell densities higher than usually seen in these media, but the yield of active ADH product was poor.
The optimal glucose feeding rate is dependent on the efficiency of aeration. In EnPresso cultures glucose release rate can be easily adjusted by the concentration of the EnZ I’m to be lower in Erlenmeyer flasks and higher in the highly aerated Ultra Yield Flasks.
Maciej Pilarek and coauthors presented his work on enhanced plasmid production using the EnBase technology and perfluorodecalin (PFD). This poster was presented at the IV Congress of Polish Biotechnology and IV EUROBIOTECH 2011 in Krakow, Poland.
This work shows the added benefit of using EnBase Flo and providing oxygen enriched PFD in microscal cultures. The two elements together provided 50% higher concentration of pDNA and the amount of plasmid copies per cell was higher. This provides new opportunities to perform increased plasmid protduciton in microscale cultivations in microscale cultrues within 8 hours.
Eva Brand - International Biotechnology Symposium and Exhibition - Rimini, Italy September 14-18, 2010
This cultivation strategy for plasmid production is faster and more simple than standard plasmid production, is applicable to be incorporated in plasmid preparation kits, and is applicable to automation and high throughput approaches.
With the novel EnBase® technology it is possible to cultivate E. coli to higher cell densities even at very small scales due to an internal fed‐batch system. This is the prerequisite also for plasmid production in small scale. Here we present results for a laboratory application of EnBase®, where picked bacteria (e.g. from a transformation plate) can grow in 50 μL EnBase® culture in a closed 1.5 mL reaction tube on a thermomixer without any requirement for an extra device. Furthermore, plasmid production with EnBase is possible in high throughput applications in 96‐well plates with 2‐5 μg plasmid per well.
Successful screening for microbial whole cell biotransformations strongly depends on the metabolic state of the cells. Furthermore screening is simplified by higher cell densities which provide the possibility to reduce the cultivation volume and scale down the cultiation process to microwell format. Thus the realization of a robust physiological state and simple high cell density cultivation in microplates in parallel is a key issue in this area. In this study we applied a microscale fed-batch technique, EnBase®[1], for the growth of yeast cultures in 24 deepwell plates for the enantioselective biotransformation of 2-butanone to R/S-2-butanol. The Screening of biocatalytic activity was carried out in a screening system with a direct head-space measurement for the chiral gas chromatography analysis. In this MPS-BioScreen System the growth of the biomass, the Biotransformation and the direct measurement of biocatalytic samples could be combined.
Conclusions:
Kaisa Ukkonen presented at the 5th Japan–Finland Biotechnology Symposium on June 8th, 2010, in Turku, Finland.
The EnBase® medium has now been developed into a tablet format, called EnPresso™, to provide an effortless and convenient procedure for successful recombinant protein production, often in significantly lower culture volumes than typically required for sufficient protein yield in conventional media.
The EnPresso™ tablets provide:
Julia Glazyrina
The previous EnBase® system was characterized by a 3-phase system, a storage gel containing the polymer, a gel for controlling the release to the medium, and the overlaying liquid medium. Although this set up is advantageous by providing the possibility to supply high amounts of substrate, its preparation is laborious and its application is limited to small volumes. Here we present the advanced EnBase® Flo system with a liquid polysaccharide serving as a basic substrate.
Conclusions EnBase® is an excellent tool for high cell density cultivations of E. coli and recombinant protein expression. The EnBase® technology is scalable from small scale cultivations in plates up to large scale fermentations. EnBase® cultivations can improve the formation of soluble protein due to controlled cell growth. The addition of medium supplements to small scale cultivations or of amylase during fermentations increases the cell growth .
BioSilta Oy FI-90014 Oulu, Finland T: +358-8-5532361 F: +358-207-001500 E: info@biosilta.com
Copyright © 2010 BioSilta Oy; All rights reserved. Prices do not include VAT.