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Abstracts / Journal of Biotechnology 131S (2007) S98–S121 S107 References Appel, D., Lutz-Wahl, S., Fischer, P., Schwaneberg, U., Schmid, R.D., 2001.A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and het-eroarenes. J. Biotechnol. 88, 167–171.Mouri, T., Michizoe, J., Ichinose, H., Kamiya, N., Goto, M., 2006. A recom-binant Escherichia coli whole cell biocatalyst harboring a cytochromeP450cam monooxygenase system coupled with enzymatic cofactor regen-eration. Appl. Microbiol. Biotechnol. 72, 514–520.Schrader, J., Berger, R.G., 2001. Biotechnology 10. Wiley-VCH, pp. 373–422. doi:10.1016/j.jbiotec.2007.07.183 16.On the transfructosylation activity and selectivity of microbial beta-fructofuranosidases for the production of prebiotics Lucia Fernandez-Arrojo a , Miguel Alvaro b , Iraj Ghazi a ,Miguel De Abreu b , Dolores Linde b , Patricia Gutierrez-Alonso b , Miguel Alcalde a , Jesus Jimenez-Barbero c , AntonioJimenez b , Antonio Ballesteros a , Maria Fernandez-Lobato b ,Francisco J. Plou a , ∗ a Instituto De Catalisis - CSIC, Marie CURIE 2, Cantoblanco,28049 Madrid, Spain b Centro de Biolog´ıa Molecular Severo Ochoa, UAM-CSIC, Madrid, Spain c Centro de Investigaciones Biol´ ogicas, CSIC, Instituto DeCatalisis-CSIC,MarieCURIE2,Cantoblanco,28049Madrid,Spain Fructooligosaccharides of the inulin-type ( 1 F-FOS) are fruc-tose oligomers with 2-4 fructosyl moieties (2 → 1)-linked,with the last one -linked to a -glucosyl group, e.g. 1-kestose(GF 2 ),nystose(GF 3 ). 1 F-FOSarewidelyusedinthefoodindus-try due to their prebiotic properties (i.e. they are selectivelyfermented by beneficial colonic flora such as Bifidobacterium and Lactobacillus ). Other fructooligosaccharides are excellentcarbohydrate sources for growth of bifidobacteria, and mayhave enhanced prebiotic effects compared with those of com-mercial 1 F-FOS: (1) neo-fructooligosaccharides (neo-FOS or 6 G-FOS), e.g. neo-kestose (neo-GF2) and neo-nystose (neo-GF3),inwhichafructosylunitis (2 → 6)-linkedtotheglucosylmoiety of sucrose and 1-kestose, respectively; (2) 6 F-FOS, suchas6-kestoseand6-nystose,inwhichfructosylunitsare (2 → 6)bound, with the last one -linked to a -glucosyl group.FOS are produced through fructosyl transfer from sucroseusingafungal -fructofuranosidase(EC3.2.1.26)orafructosyl-transferases(EC2.4.1.9).TheFOSyielddependsontherelativerates of the competing transfructosylation and hydrolysis reac-tions. Thus, some enzymes exhibit a marked preference fortransglycosylation whereas for others the hydrolysis of sucroseis the preferred process.We have purified a fructosyltransferase from Aspergillusaculeatus present in commercial samples of Pectinex UltraSP-L (Ghazi et al., 2007). We have also isolated and purifiedextracellular -fructofuranosidases from cultures of the yeasts Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) (Fern´andez-Lobato et al., 2005), Schwanniomyces occidentalis (Alvaro-Benito et al., 2007) and Rhodotorula gracilis. Thetransferase activity of these enzymes was analysed in detail,along with that of -fructofuranosidase from Saccharomycescerevisiae. Interestingly, the maximum FOS yield (expressedas weight percentage of the total amount of carbohydrates inthe mixture) was significantly higher with the enzyme from A. aculeatus (61%), compared with those from X. dendror-hous , S. occidentalis and R. gracilis (17–19%), and that from S. cerevisiae (8%). In addition, significant differences wereencountered in the selectivity of the process: A. aculeatus synthesizedonly 1 F-FOS, X.dendrorhous formedbasicallyneo-kestose , whereas S.occidentalis and S.cerevisiae wereselectivetowards the production of 6-kestose. R. gracilis synthesized acomplex mixture of di-, tri- and tetrasaccharides. The structuralfeatures that determine the transferase to hydrolase ratio as wellas the selectivity in the glycoside hydrolase family 32 are notstill well understood. Acknowledgments ThisworkwassupportedbyagrantfromtheSpanishMinistryof Education and Science (BIO2004-03773-C04), by GenomaEspa˜na (the National Foundation for Promoting Genomics andProteomics), and by an institutional grant from the Fundaci´onRam´on Areces to the Centro de Biolog´ıa Molecular SeveroOchoa References Alvaro-Benito, M., de Abreu, M., Fernandez-Arrojo, L., Plou, F.J.,Jim´enez-Barbero, J., Ballesteros, A., Polaina, J., Fern´andez-Lobato, M.,“Characterization of a -fructofuranosidase from Schwanniomyces occi-dentalis with transfructosylating activity yielding the prebiotic 6-kestose”. J. Biotechnol. , accepted (2007). Corresponding to Spanish Patent ES-200503195. PCT application submitted in Dec/2006.Fern´andez-Lobato, M., Mac´ıas, I., Mar´ın, D., Linde, D., Fern´andez-Arrojo, L.,Plou, F.J., “Novel enzyme, with fructofuranosidase activity, for the prepara-tion of prebiotic oligosaccharides”. Patent PCT/ES2006/000435 (2005).Ghazi, I., Fern´andez-Arrojo, L., Garc´ıa-Arellano, H., Ferrer, M., Plou, F.J.,Ballesteros, A., 2007. Purification and kinetic characterization of a fruc-tosyltransferase from Aspergillus aculeatus . J. Biotechnol. 128, 204–211. doi:10.1016/j.jbiotec.2007.07.184 17.Mechanisms of enzymatic degradation of azo andanthraquinone dyes by bacterial CotA-laccase Luciana Pereira ∗ , Cristina Viegas, Ana Coelho, Maria PaulaRobalo, L´ıgia Martins ITQB, 12-2781-901 Lisboa, Portugal Enzymatic bioremediation has become an attractive alterna-tive to support the bio-treatment techniques currently availableas enzymes provide simpler systems than a whole organismand could be improved by the use of modern engineeringtechniques by using rational (site-directed mutagenesis), semi-rational(i.e.combinatorialsaturationmutagenesis)anddirectedevolution approaches. Laccases constitute a large subfamily S108 Abstracts / Journal of Biotechnology 131S (2007) S98–S121 of multicopper oxidases and have a great potential in variousbiotechnological processes mainly due to their high relativenon-specific oxidation capacity, the lack of a requirement forcofactors, and the use of readily available oxygen as an electronacceptor.Fungal laccases have been confirmed for its ability todecolouriseawidenumberofazoandantraquinonedyes.Thesedyes account for more than 50% of all dyes in the textile,food, pharmaceutical, leather, cosmetics and paper industriesand therefore, azo dyes are the most common synthetic col-orants released into the environment. Colour removal fromwastewaters with traditional physical-chemical processes, suchas coagulation, adsorption and oxidation with ozone are unableto degrade all dyes currently used and these compounds aregenerally resistant to degradation by biological treatment meth-ods. Furthermore, some of the aromatic amines generated thesebiological processes are toxic and carcinogenic.In this study, enzymatic degradation of the azo dye SudanOrange and of the antraquinone dye Acid Blue 62 was exam-ined. The CotA-laccase is a bacterial laccase from Bacillussubtilis that has been extensively studied at the molecular level(Martins et al., 2002; Enguita et al., 2003; Dur˜ao et al., 2006).The main objectives of such studies are to dissect the catalyticmechanisms and to design laccases that better match biotech-nological applications, using protein engineering techniques.Only little information is available about the reactivity of azoand anthraquinonic dyes and the nature of their degradationproducts. Moreover, their products may be more toxic than theoriginalcompound.Thekineticsofazoandanthraquinonicdyesoxidation was studied and the main products were separated byHPLC and identified by NMR and MS. The possible mecha-nisms involved in the degradation are discussed. Finally, wehave examined the toxicity of the degradation products. References Dur˜ao, P., Bento, I., Fernandes, A.T., Melo, E.P., Lindley, P.F., Martins, L.O.,2006. Perturbations of the T1 copper site in the CotA laccase from Bacillussubtilis: structural, biochemical, enzymatic and stability studies. J. Biol.Inorg. Chem. 11, 514–26526.Enguita, F.J., Martins, L.M., Henriques, A.O., Carrondo, M.A., 2003. Crystalstructure of a bacterial endospore coat component. A laccase with enhancedthermostability properties. J. Biol. Chem. 278, 19416–19425.Martins, L.O., Soares, C.M., Pereira, M.M., Teixeira, M., Jones, G.H., Hen-riques, A.O., 2002. Molecular and biochemical characterization of a highlystable bacterial laccase that occurs as a structural component of the Bacillussubtilis endospore coat. J. Biol. Chem. 277, 18849–18859. doi:10.1016/j.jbiotec.2007.07.185 18.Insight into stability of multi-copper oxidases Andre T. Fernandes a , ∗ , L´ıgia O. Martins a , Eduardo P. Melo ba ITQB, Av da Republica (EAN), 2781-901 Oeiras, Portugal b Bioengineering, Centre for Molecular and Structural Biomedicine, Universidade do Algarve, Faro, Portugal The multi-copper oxidases (MCOs) constitute a family of enzymes whose principal members are ceruloplasmin (Fe(II)oxygen oxidoreductase, EC 1.16.3.1), ascorbate oxidase (L-ascorbate oxygen oxidoreductase, EC 1.10.3.3) and laccase(benzenediol oxygen oxidoreductase, EC 1.10.3.2) (Stoj andKosman, 2005). MCO’s have a broad substrate specificity, andoxidise numerous aromatic phenols and amines reducing themolecular oxygen to water upon receipt of four electrons. Onlya few members of this family present higher specificity to lowervalent metal ions such as Mn 2+ , Fe 2+ or Cu + , being thus desig-nated as metallo-oxidases (Stoj and Kosman, 2005). Laccasesare potential biocatalysis for diverse biotechnology applica-tions mainly due to their high relative non-specific oxidationcapacity, the lack of requirement for cofactors, and the use of readily available oxygen as an electron acceptor. Owing to lac-casesbiotechnologypotential,understandingthestabilizationof enzymes from hyperthermophilic organisms is also critical fordesigning efficient enzymes that can work in industrial harshconditions.We had characterized the activity and stability of two mem-bers of the MCO family: the CotA laccase from Bacillus subtlis and the McoA metallo-oxidase from the hyperthermophilic Aquifex aeolicus . CotA laccase is a thermoactive and intrinsi-callythermostableenzyme.However,copperdepletionfromthetype1coppersiteisakeyeventintheinactivationoftheenzymeandthusitisadeterminantofitsthermodynamicstability(Dur˜aoet al., 2006). Attempts to increase the redox potential of CotAby site-directed mutagenesis were successful but had dramaticeffectsonthethermodynamicstabilityoftheenzyme.TheMcoAmetallo-oxidase from A. aeolicus displays cuprous and ferrousoxidase activity (Fernandes et al., 2007) and is extremely ther-mostablewithatemperatureatthemid-pointrangingfrom105to114 ◦ C(Fernandesetal.,submitted).Enhancedthermalstabilityof McoA relies on a flat dependence of stability on tempera-ture with a low stability at room temperature (2.8kcal/mol) butextending for a large range of temperatures. In both proteinscopper ions are crucial in the stability of their tertiary structure. References Stoj,C.S.,Kosman,D.J.,2005.Cooperproteins:Oxidases.In:King,R.B.(Ed.),Encyclopedia of Inorganic Chemistry, Vol. II, 2nd ed. John Wiley & Sons,pp. 1134–1159.Durao, P., Bento, I., Fernandes, A.T., Melo, E.P., Lindley, P.F., Martins, L.O.,2006. Perturbations of the T1 copper site in the CotA laccase from Bacillussubtilis : structural, biochemical, enzymatic and stability studies. J. Biol.Inorg. Chem. 11, 514–526.Fernandes, A.T., Soares, C.M., Pereira, M.M., Huber, R., Grass, G., Martins,L.O., 2007. FEBS J. In Press . Fernandes, AT, Martins LO, Melo, EP (2007) submitted . doi:10.1016/j.jbiotec.2007.07.186
