New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) Orals Sunday 13 July References Industrial biotechnology from fundamentals [1].Tsuji Y, Fujihara T. Chem Commun 2012;48:2365. [2].Glueck SM, Gümüs S, Fabian WMF, Faber K. Chem Soc Rev to practice (ACIB Session) 2010;39:313. [3].Matsui T, Yoshida T, Yoshimura T, Nagasawa T. Appl Microbiol Bio- ACIB-1 technol 2006;73:95. [4].Lindsey AS, Jeskey H. Chem Rev 1957;57:583. Two promising biocatalytic tools: regioselective car- [5].Wuensch C, Gross J, Steinkellner G, Lyskowski A, Gruber K, Glueck boxylation of aromatics and asymmetric hydration of SM, Faber K. RSC Adv 2014;4:9673. alkenes [6].Wuensch C, Gross J, Steinkellner G, Gruber K, Glueck SM, Faber K. Angew Chem Int Ed 2013;52:2293. Silvia M. Glueck 1,∗ , Christiane Wuensch 1, Tamara Reiter 1, http://dx.doi.org/10.1016/j.nbt.2014.05.1615 Johannes Gross 1, Georg Steinkellner 1, Andrzej Lyskowski 1, Karl Gruber 2, Kurt Faber 2 1 ACIB GmbH c/o University of Graz, Department of Chemistry, Austria ACIB-2 2 University of Graz, Austria Unconventional substrates for enzymatic reduction: car- Due to the growing demand for alternative carbon sources, boxylates and nitriles the development of CO2-fixation strategies for the production M. Winkler 1,∗ K. Napora-Wijata 1 B. Wilding 1 N. Klempier 2 of valuable chemicals is a current challenge in synthetic organic , , , chemistry [1]. The enzymatic carboxylation of aromatic com- 1 ACIB GmbH, Petersgasse 14/III, 8010 Graz, Austria 2 pounds [2,3] catalyzed by various decarboxylases represents a Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse promising ‘green’ alternative to the classic Kolbe-Schmitt reaction 9, 8010 Graz, Austria [4] which requires harsh reaction conditions. Depending on the type of enzyme, the carboxylation proceeded Keywords: Biocatalysis; Nitrile Reductase; Carboxylate reductase; in a highly regio-complementary fashion: Benzoic acid decarboxy- Reduction; Enzyme lases selectively catalyzed the ortho-carboxylation of phenolic substrates (conv. up to 80%) whereas phenolic acid decarboxylases The reduction of carbonyls by enzymes has been extensively exclusively catalyzed the ␤-carboxylation of styrenes (conv. up to studied and is now widely applied on industrial scale. In com- 40%) [5]. parison, enzyme catalyzed carboxylic acid/carboxylate and nitrile During these studies a promiscuous ‘hydratase-activity’ of phe- reduction are relatively young fields. Their full potential is cur- nolic acid decarboxylases was discovered, which leads to the rently being explored. The chemical reduction of nitriles and stereoselective hydration of hydroxystyrenes to yield the corre- carboxylic acids requires strong reducing agents due the low reac- sponding sec-alcohols with high conversion (up to 82%) and e.e.’s tivity of these moieties. Consequently, mild alternatives would be of up to 71% [6]. a valuable addition to the current toolbox of biocatalysis. The Acknowledgements: This work has been supported by the products of nitrile reduction are primary amines and we have Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT recently expressed nitrile reductase enzymes (NReds), in order to through the Austrian FFG-COMET-Funding Program. apply these enzymes as biocatalysts. The substrate scopes of wild type and mutant NReds as well as their biochemical characteristics were studied [1]. The reaction mechanism of NReds involves the 1871-6784/$ — see front matter www.elsevier.com/locate/nbt S1 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) New Biotechnology · Volume 31S · July 2014 Acknowledgement: This project is financed by the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 289646 (Kyrobio). References [1].Winkler M, et al. Comprehensive chirality. Synthetic methods VI, vol. 7. Amsterdam: Elsevier; 2012. p. 350–71. [2].Hajnal I, et al. FEBS J 2013;280:5815–28. Figure 1 Whole cell biotransformation approach towards 3- http://dx.doi.org/10.1016/j.nbt.2014.05.1617 hydroxytyrosol – an antioxidant from olives. reaction of the substrate with an active site thiol and subsequent ACIB-4 NADPH mediated reduction of the intermediate [2], which is simi- Enzyme responsive polymers lar to the mechanism proposed for carboxylate reductase enzymes (CARs) [3]. The latter enzyme was in focus of the development of a Alexandra Rollett 1,∗ , Andrea Heinzle 2, Konstantin Schneider 2, whole cell system (Figure 1) for the production of the antioxidant Doris Schiffer 2, Gregor Tegl 3, Eva Sigl 2, Georg Guebitz 1 3-hydroxytyrosol [4]. 1 BOKU-Vienna/ACIB GmbH, Austria 2 ACIB-Austrian Centre of Industrial Biotechnology GmbH, Austria References 3 BOKU-Vienna, Austria [1].(a) Wilding B, et al. Adv Syn Catal 2012;354(11–12):2191–8; (b) Wilding B, et al. Chem Eur J 2013;19(22):7007–12. Smart polymers can change their properties depending on [2].Chikwana VM, et al. J Biol Chem 2012;387:30560–70. certain stimuli like pH, temperature, light or electrical fields. Espe- [3].Venkitasubramanian P, et al. J Biol Chem 2007;282:478–85. cially in biomedical materials, but also in technical applications, [4].Napora-Wijata K, et al. ChemCatChem, Patent application pending [in print]. like the packaging industry, enzymes are useful biomarkers for sensoring purposes. Here we present different strategies towards http://dx.doi.org/10.1016/j.nbt.2014.05.1616 enzyme responsive polymer systems for the detection of microbial contaminations in distinctive fields. Enzyme responsive hydrogel based systems were constructed ACIB-3 using methacrylated biopolymers like polygalacturonic acid [1], alginic acid [2], carboxymethylcellulose [3], peptidoglycan, Engineering of cupin hydroxynitrile lyases gelatin, collagen, alginate and agarose [4–6] while both stabil- ,∗ Kerstin Steiner 1 , Romana Wiedner 1, Bettina Kothbauer 1, Man- ity and sensitivity were tuned via the degree of UV crosslinking. dana Gruber-Khadjawi 1, Helmut Schwab 2 Theses systems were successfully used to detect contaminating 1 ACIB GmbH, Austria microorganisms based on their extracellular enzymes, and infec- 2 TU Graz, Austria tion of wounds based on enzymes from the human immune system. The latter included myeloperoxidase, cathepsin G, elas- Enantiopure cyanohydrins serve as versatile building blocks for tase and lysozyme, which were found to show an elevated activity a broad range of chemical and enzymatic reactions resulting in in infected wounds. highly valuable products for many applications. Hydroxynitrile References lyases comprise a diverse group of enzymes which catalyze the reversible cleavage of cyanohydrins to carbonyl compounds and [1].Schneider, et al. N Biotechnol 2012;29:502–9. [2].Schneider, et al. Enzyme Microbial Technol 2011;48:312–8. hydrogen cyanide [1]. For a long time HNLs were only known to [3].Schneider, et al. Process Biochem 2012;47:305–11. exist in plants, but recently (R)-selective HNL activity has been [4].Hasmann, et al. Exp Dermatol 2011;20:508–13. identified in several bacterial proteins [2]. The structure of one [5].Heinzle, et al. Wound Repair Regen 2013;21:482–9. target protein from Granulicella tundricola (GtHNL) was solved and [6].Hasmann, et al. Diag Microbiol Infect Dis 2011;71:12–23. showed high similarity to proteins of the cupin superfamily, a fold, http://dx.doi.org/10.1016/j.nbt.2014.05.1618 which has not been reported for HNLs before. Cupins are ubiqui- tous small beta-barrel proteins and ideal candidates for application in industrial processes and as scaffold for enzyme engineering as they are easily expressed as soluble proteins in exceptionally high yield (> 50% of total protein) in E. coli. A detailed bio- chemical characterisation of GtHNL revealed that the enzyme is metal-dependent. Several amino acids were identified, which are important for activity. The initial specific activity of 1.74 U/mg at pH 6 with (R)-mandelonitrile, and the 80% conversion of benzal- dehyde to (R)-mandelonitrile with 90% enantiomeric excess were significantly improved by applying random and rational protein engineering approaches. S2 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) ACIB-5 ACIB-6 Esterases from Clostridium are involved in anaerobic Designing robust Saccharomyces cerevisiae strains degradation of synthetic polyester against stresses encountered during bioethanol fermen- tations from lignocellulosic biomass Veronika Perz 1,∗ , Veronika Perz 2, Armin Baumschlager 2, Klaus Bleymaier 2, Andrzej Łyskowski 2, Altijana Hromic 2, Karl Gruber 3, Vinod Kumar 1,∗ , Darren Greetham 1, Tithira Wimalasena 2 4 4 4 2 Carsten Sinkel , Ulf Küper , Melanie Bonnekessel , Doris Ribitsch , 1 The University of Nottingham, United Kingdom 5 Georg Guebitz 2 Kingston Research Limited (BP and DuPont JV), United Kingdom 1 ACIB GmbH, Austria 2 ACIB GmbH, Petersgasse 14, 8010 Graz, Austria During the pre-treatment of lignocellulosic biomass inhibitory 3 Institute of Molecular Biosciences, University of Graz, Austria compounds are released which can exert adverse effects on cellular 4 BASF SE, Carl-Bosch-Straße 38, 67056 Ludwigshafen, Germany growth, metabolism and ethanol production. In addition, osmotic 5 Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Austria stress caused by the high concentrations of available sugars and end-stage ethanol toxicity