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Banning heavy metals from paints: Enzymatic hardening of alkyd resins
ABSTRACTS
New Biotechnology · Volume 29S · September 2012
Banning heavy metals from paints: Enzymatic hardening of alkyd resins K. Greimel 1 , V. Perz 1 , E. Herrero Acero 1 , G. Gübitz 1,2 1
Austrian Centre of Industrial Biotechnology ACIB, Graz, Austria Institute of Environmental Biotechnology, Graz University of Technology, Austria 2
Alkyd resins are a common type of paints containing unsaturated fatty acids. During the hardening process the unsaturated groups are chemically oxidized and consequently cross-linked. This process is accelerated by different types of metal complexes, whereas cobalt based complexes are the most effective ones. Environmental and health awareness of the market is forcing the coating industry towards the use of environmentally friendlier siccatives due to the fact that cobalt catalysts are suspected to be potential carcinogenic in aerosol [1,2]. The optimal solution for drying alkyd resins would comprise the complete replacement of currently used heavy metals by a non-toxic and environmentally friendly biocatalyst. In this work we present for the first time an enzyme based drying system for the curing of alkyd resins. The potential of a laccase from Trametes hirsuta in combination with two different electron mediators was evaluated regarding its capability to crosslink the unsaturated fatty acids and consequently harden the resin. Measurements of the amount of oxygen consumed by the laccase during the reaction progress allowed to compare the effectiveness of the different mediators. The measurable decrease in air saturation after the addition of alkyd resin to the preoxidized mediator was correlated to the extent of the oxidation (Figure 1).
Figure 1 Oxygen consumption during cross-linking. Size exclusion chromatography was used to characterize the molecular weight increase. A 35% increase was observed after a reaction time of 3 h in the laccase/mediator treated sample. A further step was to perform experiments with films mimicking a drying coating. The drying reaction of alkyd resin treated with cobalt, ThL/ABTS and ThL/HBT, respectively was monitored with FTIR spectroscopy. A clear decrease of the band assigned to double bonds (3010 cm−1 ) was correlated to the drying of the alkyd resin. Finally, we successfully demonstrated the capability of the
Figure 2 Characterization of the drying reaction using FTIR spectroscopy (1: resin, 2: resin + Co, 3: resin + ThL + ABTS, 4: resin + ThL + HBT). S238
www.elsevier.com/locate/nbt
New Biotechnology · Volume 29S · September 2012
ABSTRACTS
laccase/mediator system to harden alkyd resins, using drying recorder measurements. Without the laccase/mediator system, no drying was observed within 2 days. When using our developed system, complete drying was achieved after only 43 h (Figure 2).
Novel human kidney cell lines for better prediction of nephrotoxicity
References
1
1. Bucher JR, et al. Toxicol Sci 1999;49(1):56–67. 2. Lison D, et al. Occup Environ Med 2001;58(10):619–62.
http://dx.doi.org/10.1016/j.nbt.2012.05.012 Engineering by design: Systems biology based development of biotechnological production strains Diethard Mattanovich 1,2 1 2
University of Natural Resources and Life Sciences Vienna, Austria Austrian Centre of Industrial Biotechnology, Vienna, Austria
For the development of efficient production strains it is crucial to understand the molecular physiology of the host, and the specific limitations that the product may exert upon production. Systems biology is a powerful tool to obtain a comprehensive knowledge base, leading to the identification of key limiting factors, which can subsequently be applied for the design and construction of improved production platforms. Research on cell engineering in the ACIB laboratories focuses on rational development of microbial and animal cell hosts for the production of biochemicals, industrial enzymes and biopharmaceutical proteins. The common frame is the application of systems biotechnology methods for a comprehensive understanding of host physiology and production processes, further applied for the targeted development of superior production platforms. After an overview on bacterial, fungal and mammalian cell engineering, the implementation of systems biotechnology to improve protein folding and secretion in yeast will be discussed. Transcriptomic, proteomic and metabolomic data are applied in a coordinated fashion to identify candidate genes for strain engineering. Their manipulation enhanced protein productivity several fold. This research, combined with biochemical analysis of the folding and secretion pathway gave insight to cellular bottlenecks as engineering targets. Additionally, the design of an improved profile of the correlation of protein secretion vs. cell growth was realized by engineering the cell cycle, and resulted in higher product titers as more cells were kept in the more productive phases of the cell cycle. It will be discussed how these systems biology approaches lead to a detailed understanding of host cell physiology during product formation to apply them to design and construct improved production processes.
Lukas Fliedl 1 , Jens Jennings 4 , Matthias Grillari-Voglauer 1,2,3
Pontiller 2 , Sarah Wieser 1 , Johannes
Dunzinger 2,3 , Paul Grillari 2,3 , Regina
Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria 2 Evercyte GmbH, Muthgasse 18, 1190 Vienna, Austria 3 Institute of Applied Microbiology, Department of Biotechnology, BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Austria 4 Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria Over the last two decades in vitro model systems have proven necessary to predict toxic effects caused by drugs or chemicals, whereby the choice of the cell model used is decisive for the quality of the prediction. For example LLC-PK1, a porcine kidney cell line, is routinely used in drug-induced nephrotoxicity studies, although interspecies variations hamper the potential of this cell line in predicting toxic effects on humans. Alternatively, primary human renal proximal cells are used to study toxicity, but the use of normal cells is limited due to the restricted number of population doublings reached in vitro. HK-2, a continuously growing human kidney cell line immortalised by introduction of human papilloma virus genes, was suggested as an alternative, because of its human origin. However, this cell line shows limited comparability to primary cells. Therefore, we have recently established a human proximal tubular epithelial cell line RPTEC/TERT1 by introduction of the catalytic subunit of human telomerase, showing all characteristics of the corresponding normal cells. Now we have taken this one step further by establishing the same cell type from human urine (HEPTEC) bearing the opportunity to non-invasively establish renal cell lines from any consenting individual independent of age, sex, disease type or status. Furthermore, we have generated a new human renal proximal tubular epithelial cell line by the introduction of the viral oncogenes SV40 large T/small t. These cell models enable the comparison of the effects of the mentioned immortalisation methods on the maintenance of cell type specific markers and characteristics. To show the applicability of our model systems we tested agents such as cisplatin known for their nephrotoxic effects. The observed results prove the advantages of our cell model systems over currently used ones for toxicity studies. Moreover due to the fact that HEPTECs can be derived non-invasively from any person, these model systems can bring new relevance to the area of toxicogenetics. http://dx.doi.org/10.1016/j.nbt.2012.05.014
http://dx.doi.org/10.1016/j.nbt.2012.05.013
www.elsevier.com/locate/nbt S239
New Biotechnology · Volume 29S · September 2012
Banning heavy metals from paints: Enzymatic hardening of alkyd resins K. Greimel 1 , V. Perz 1 , E. Herrero Acero 1 , G. Gübitz 1,2 1
Austrian Centre of Industrial Biotechnology ACIB, Graz, Austria Institute of Environmental Biotechnology, Graz University of Technology, Austria 2
Alkyd resins are a common type of paints containing unsaturated fatty acids. During the hardening process the unsaturated groups are chemically oxidized and consequently cross-linked. This process is accelerated by different types of metal complexes, whereas cobalt based complexes are the most effective ones. Environmental and health awareness of the market is forcing the coating industry towards the use of environmentally friendlier siccatives due to the fact that cobalt catalysts are suspected to be potential carcinogenic in aerosol [1,2]. The optimal solution for drying alkyd resins would comprise the complete replacement of currently used heavy metals by a non-toxic and environmentally friendly biocatalyst. In this work we present for the first time an enzyme based drying system for the curing of alkyd resins. The potential of a laccase from Trametes hirsuta in combination with two different electron mediators was evaluated regarding its capability to crosslink the unsaturated fatty acids and consequently harden the resin. Measurements of the amount of oxygen consumed by the laccase during the reaction progress allowed to compare the effectiveness of the different mediators. The measurable decrease in air saturation after the addition of alkyd resin to the preoxidized mediator was correlated to the extent of the oxidation (Figure 1).
Figure 1 Oxygen consumption during cross-linking. Size exclusion chromatography was used to characterize the molecular weight increase. A 35% increase was observed after a reaction time of 3 h in the laccase/mediator treated sample. A further step was to perform experiments with films mimicking a drying coating. The drying reaction of alkyd resin treated with cobalt, ThL/ABTS and ThL/HBT, respectively was monitored with FTIR spectroscopy. A clear decrease of the band assigned to double bonds (3010 cm−1 ) was correlated to the drying of the alkyd resin. Finally, we successfully demonstrated the capability of the
Figure 2 Characterization of the drying reaction using FTIR spectroscopy (1: resin, 2: resin + Co, 3: resin + ThL + ABTS, 4: resin + ThL + HBT). S238
www.elsevier.com/locate/nbt
New Biotechnology · Volume 29S · September 2012
ABSTRACTS
laccase/mediator system to harden alkyd resins, using drying recorder measurements. Without the laccase/mediator system, no drying was observed within 2 days. When using our developed system, complete drying was achieved after only 43 h (Figure 2).
Novel human kidney cell lines for better prediction of nephrotoxicity
References
1
1. Bucher JR, et al. Toxicol Sci 1999;49(1):56–67. 2. Lison D, et al. Occup Environ Med 2001;58(10):619–62.
http://dx.doi.org/10.1016/j.nbt.2012.05.012 Engineering by design: Systems biology based development of biotechnological production strains Diethard Mattanovich 1,2 1 2
University of Natural Resources and Life Sciences Vienna, Austria Austrian Centre of Industrial Biotechnology, Vienna, Austria
For the development of efficient production strains it is crucial to understand the molecular physiology of the host, and the specific limitations that the product may exert upon production. Systems biology is a powerful tool to obtain a comprehensive knowledge base, leading to the identification of key limiting factors, which can subsequently be applied for the design and construction of improved production platforms. Research on cell engineering in the ACIB laboratories focuses on rational development of microbial and animal cell hosts for the production of biochemicals, industrial enzymes and biopharmaceutical proteins. The common frame is the application of systems biotechnology methods for a comprehensive understanding of host physiology and production processes, further applied for the targeted development of superior production platforms. After an overview on bacterial, fungal and mammalian cell engineering, the implementation of systems biotechnology to improve protein folding and secretion in yeast will be discussed. Transcriptomic, proteomic and metabolomic data are applied in a coordinated fashion to identify candidate genes for strain engineering. Their manipulation enhanced protein productivity several fold. This research, combined with biochemical analysis of the folding and secretion pathway gave insight to cellular bottlenecks as engineering targets. Additionally, the design of an improved profile of the correlation of protein secretion vs. cell growth was realized by engineering the cell cycle, and resulted in higher product titers as more cells were kept in the more productive phases of the cell cycle. It will be discussed how these systems biology approaches lead to a detailed understanding of host cell physiology during product formation to apply them to design and construct improved production processes.
Lukas Fliedl 1 , Jens Jennings 4 , Matthias Grillari-Voglauer 1,2,3
Pontiller 2 , Sarah Wieser 1 , Johannes
Dunzinger 2,3 , Paul Grillari 2,3 , Regina
Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria 2 Evercyte GmbH, Muthgasse 18, 1190 Vienna, Austria 3 Institute of Applied Microbiology, Department of Biotechnology, BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Austria 4 Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria Over the last two decades in vitro model systems have proven necessary to predict toxic effects caused by drugs or chemicals, whereby the choice of the cell model used is decisive for the quality of the prediction. For example LLC-PK1, a porcine kidney cell line, is routinely used in drug-induced nephrotoxicity studies, although interspecies variations hamper the potential of this cell line in predicting toxic effects on humans. Alternatively, primary human renal proximal cells are used to study toxicity, but the use of normal cells is limited due to the restricted number of population doublings reached in vitro. HK-2, a continuously growing human kidney cell line immortalised by introduction of human papilloma virus genes, was suggested as an alternative, because of its human origin. However, this cell line shows limited comparability to primary cells. Therefore, we have recently established a human proximal tubular epithelial cell line RPTEC/TERT1 by introduction of the catalytic subunit of human telomerase, showing all characteristics of the corresponding normal cells. Now we have taken this one step further by establishing the same cell type from human urine (HEPTEC) bearing the opportunity to non-invasively establish renal cell lines from any consenting individual independent of age, sex, disease type or status. Furthermore, we have generated a new human renal proximal tubular epithelial cell line by the introduction of the viral oncogenes SV40 large T/small t. These cell models enable the comparison of the effects of the mentioned immortalisation methods on the maintenance of cell type specific markers and characteristics. To show the applicability of our model systems we tested agents such as cisplatin known for their nephrotoxic effects. The observed results prove the advantages of our cell model systems over currently used ones for toxicity studies. Moreover due to the fact that HEPTECs can be derived non-invasively from any person, these model systems can bring new relevance to the area of toxicogenetics. http://dx.doi.org/10.1016/j.nbt.2012.05.014
http://dx.doi.org/10.1016/j.nbt.2012.05.013
www.elsevier.com/locate/nbt S239