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leads to reducing sugars (ramnose and glucose), to prunin and to the aglycone, naringenin. These flavonoids have some interesting activities. Naringenin has shown anti-oxidant, antiulcer, anti-mutagenic and anti-inflammatory activity, as well as antiproliferative effects and inhibition of aflotoxin B1-induced carcinogenesis, inhibiting the proliferation of breast cancer and delaying mammary tumorigenesis and can also function as chemopreventive agent against neurodegenerative diseases such as Alzheimer’s disease. The main goal of this study was to maximize naringenin production through naringin enzymatic hydrolysis by naringinase. One of the problems of this enzymatic reaction is the low solubility of naringin in aqueous media (1.96 g/L, 45 ◦ C). To overcome this low solubility of naringin the effect of different solvents was studied. In order to develop a biphasic reaction system, for naringin hydrolysis by naringinase, a selection of different solvents was carried out according to their log P values and functionality. Thus, octane and n-hexane were representative of unbranched alkanes; cyclohexane of cyclic alkanes; 1-octanol, 2-octanol and 1-hexanol of alcohols; toluene and anisole of aromatic compounds; ethylic ether of ethers; ethyl acetate of esters; carbon tetrachloride and chloroform of halogenated solvents. The limitation to the development of these systems was the low solubility of naringin in this kind of non-polar solvents which had excluded the biphasic design system. To enhance naringin concentration in the enzymatic reaction media some solvents were tested in water co-solvation systems according to its relative permittivity, in order to maximize naringin solubilization in the aqueous system. Low solubility was observed with water, propylene carbonate, 2-propanol as well as 1-butanol. Higher solubility was obtained with DMSO, DMF, methanol, ethanol, tetrahydrofurane and 1,4-dioxane, while acetone 1,2-dimetoxiethane appear as less effective. When naringin hydrolysis by naringinase was evaluated, comparing the different co-solvent enzymatic systems, the reducing sugar concentration decreased from the reaction media with acetone to the system with DMF (acetone > 1-butanol > 2propanol > methanol > DMSO > ethanol > DMF). An increase in the initial reaction rates were observed, in the enzymatic co-solvation naringin hydrolysis system, from the reaction media with 1-butanol to 1,2-dimetoxiethane (1-butanol < 2-propanol < acetone < 1,4dioxane < tetrahydrofurane < 1,2-dimetoxiethane). doi:10.1016/j.jbiotec.2007.07.162
35. Pycnoporus sanguineus IDEA, a laccase-overproducing fungi with high potential in partial enzymatic conversion (PEC-Technology) of Venezuelan extra-heavy crude oil Hector Urbina ∗ , Angelica Reyes, Emidio Fusella, Meralys Gonzalez, Vladimir Leon, Leopoldo Naranjo Fundacion Instituto de Estudios Avanzados IDEA, Carretera Nacional Hoyo de la Puerta, Baruta, Centro de Biotecnologia, Unidad de Biotecnologia del Petr´oleo, 1080-A Caracas, Distrito Capital, Venezuela E-mail address:
[email protected]. Laccases (EC 1.10.3.2) are multicopper-containing enzymes which catalyze the oxidation of phenolic and nonphenolic compounds with the concurrent reduction of molecular oxygen to water (Otterbein et al., 2000). For this reason it has generated considerable interest as an approach to the enzymatic bleaching of Kraft pulp, delignification (Jaouani et al., 2005). Besides, laccase is an extracellular oxidative enzyme frequently used in polluted soils bioremediation and oxidation of polycyclic aromatic hydrocarbons (PAHs) (Naranjo et al., in press). However, our research team is focuses on the development of novel green processes for improvement of non-conventional hydrocarbons of the Orinoco Oil Belt from Venezuela, the biggest proven reserve of extra-heavy crude oil (EHCO) of the world. The aim of this study is to make a preliminary characterization of laccase produced by Pycnoporus sanguineus IDEA (L.) Murrill (Coriolaceae, Basiomycotina) in order to use it in partial enzymatic conversion (PEC-Technology) of EHCO (Le´on et al., 2006). P. sanguineus IDEA was found growing on a root of dicotyledonous buried in Guanoco asphalt natural lake, Sucre State (Venezuela). A little context portion of the basidiocarp was used to obtain the axenic culture, and fungal identification was carried out by morphological and molecular methods (28S rRNA gene and rDNA ITS sequences analysis). For biochemical studies, the mycelium was grown-up in Czapek minimal media supplemented by 15 g of yeast extract, and 15 g of starch per 1 L (MBYA) with or without CuSO4 as inductor (20–300 !M). The induction levels of laccase in supernatant were monitored through 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS). For cloning of laccase encoding gene of P. sanguineus IDEA, a primers pairs was designed based on laccase cDNA sequence from P. cinnabarinus (Otterbein et al., 2000). ORF1 encoded a protein with high identity to other laccases, particularly of P. cinnabarinus. For the examination of laccase activity on EHCO partial enzymatic conversion, 1.4 L supernatant (after 72 h of culture) was lyophilizated and 10 mg of cell-free powder resuspended in citrate buffer pH 5 was added to 25 g de EHCO emulsion and incubated at 30 ◦ C for 10, 20 and 30 h. The results obtained showed that the laccase activity in culture broth of P. sanguineus IDEA was improved when MBYA medium was supplemented with 150 !M of CuSO4 (1000 U/mg P) compared with control (600 U/mg P). Nevertheless, the laccase activity in this condition was incremented to 4000 U/mg P when supernatant of P. sanguineus IDEA was liophylized. The PEC was confirmed by loss of density after
Abstracts / Journal of Biotechnology 131S (2007) S74–S97
30 h demonstrating a reduction of heavy fraction contained in the EHCO, and suggesting that the complementary use of the biotechnology in the petroleum industry provides new tools to improve heavy and EHCO of the Orinoco Oil Belt from Venezuela. References Jaouani, et al., 2005. Role of Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill wastewater. Enzyme Microb. Technol. 36, 478–486. Le´on, et al., 2006. Process for the upgrading of heavy crude oil, extra-heavy crude oil or bitumens through the addition of a biocatalyst (US-Patent pending). Naranjo, et al., in press. Isolation of autochthonous non-white rot fungi with potential in Venezuelan extra-heavy crude oil bioconversion. Biocatal. Biotransfer. Otterbein, et al., 2000. Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris. Eur. J. Biochem. 267, 1619–1625.
doi:10.1016/j.jbiotec.2007.07.163 36. Reduction of redox dyes using enzymes from Bacillus subtilis and Bacillus cereus Mojca Boˇziˇc ∗ , Sina Pricelius, Georg Gubitz, Vanja Kokol University of Maribor, Faculty of Mechanical Engineering, Institute for Engineering Materials and De, 2000 Maribor, Slovenia E-mail address:
[email protected]. In the coloration of cellulose fibres, vat (including indigo) and sulphur dyes still represent a relatively large part of the dyestuff market (about 31%); among them about 120,000 t of vat dyes are being used annually. However, vat and sulphur dyes require a complicated application procedure (reduction and oxidation mechanisms) because they are practically insoluble in water and have no affinity for cellulose fibres in such a state. Thus, these dyes have to be reduced (vatted) before the dyeing to be converted into the water-soluble, i.e. leuco enolate, dye form which have substantivity towards fibres and which, after absorption into the fibres to be dyed, should be re-oxidised to the original water-insoluble dyes pigment form in situ in the fibres. The conventional method of application of these dyes is performed from a dye-bath containing sodium dithionite/sodium sulphide as reducing system representing a significant environmental problem (Boˇziˇc and Kokol, in press). A green chemistry solution for a replacement of sulfide-containing reducing agents with more environmentally friendly system would be better solution that treating/removal sulfides from such a dyeing effluent (Blackburn and Harvey, 2004). Enzymes that specifically reduce keto groups and sulphide-bonds could be an elegant alternative since they can be used under mild conditions and they do not have negative effects on environment. Several micro-organisms produce enzymes which reductively cleave the azo bond at the expense of reducing agent, typically NAD(P)H. The occurrence of these so-called azoreductases has been reported for a number of bacteria. It has been recently shown that azo-reductases from a thermoalka-
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lophilic Bacillus sp. were able to reduce a large structural variety of systematically substituted azo dyes (Pricelius et al., 2007). In this presentation it will be shown that Bacillus subtilis as well as Bacillus cereus enzymes have also capability to reduce keto groups and sulphide-bonds of selected redox dyes (indigo carmine (C.I. Acid Blue 74), Lawsone (C.I. Natural Orange 6), Diresul Negro RDT liq. (C.I. Leuco Sulphur Black 1)) in the presence of NADH. Hydrogen peroxide as an oxidizing agent will be used to oxidize the dyes into their initially forms. Acknowledgement The authors would like to thank the Slovenian Research Agency for financial support (J2-7018-0795). References Blackburn, R.S., Harvey, A., 2004. Green chemistry methods in sulfur dyeing: application of various reducing d-sugars and analysis of the importance of optimum redox potential. Environ. Sci. Technol. 38, 4034– 4039. Boˇziˇc, M., Kokol, V., in press. Ecological alternatives to the reduction and oxidation processes in dyeing with vat and sulphur dyes. Dyes Pigments. Pricelius, S., Held, C., Sollner, S., Deller, S., Murkovic, M., Ullrich, R., Hofrichter, M., Cavaco-Pauloe, A., Macheroux, P., Guebitz, G.M., 2007. Enzymatic reduction and oxidation of fibre-bound azo-dyes. Enzyme Microb. Technol. 40, 1732–1738.
doi:10.1016/j.jbiotec.2007.07.164 37. Continous asymmetric ketone reduction processes with tailor-made microorganisms Kirsten Schroer a , Eva Tacha a , Stefanie Bringer-Meyer a , Werner Hummel b , Thomas Daussmann c , Rupert Pfaller d , Stephan L¨utz a,∗ a
Research Centre J¨ulich, Institute of Biotechnology 2, Forschungszentrum J¨ulich, Leo-Brandt-Str., 52425 J¨ulich, Germany b Heinrich-Heine-University, Duesseldorf, Germany c Julich Chiral Solutions GmbH, J¨ ulich, Germany d Consortium f¨ ur Elektrochemische Industrie GmbH, Munich, Germany E-mail address:
[email protected]. Chiral compounds and especially optically active secondary alcohols are important building blocks in the chemical and pharmaceutical industry for the production of, e.g. chemical catalysts, liquid crystals, flavours, agrochemical or drugs (Daußmann et al., 2006). Alcohol dehydrogenases (ADHs), used as isolated enzymes or whole cells, catalyze the stereoselective reduction of prochiral ketones with remarkable chemo-, regioand stereoselectivity (Wandrey, 2004). In this work different types of recombinant Escherichia coli were applied as biocatalysts for the reduction of prochiral ketones to the corresponding optically active alcohols. Depending on the stereoselectivity of the recombinant ADH both enantiomeres of an optically active alcohol could be produced. By combination of the producing enzyme with suitable cofactor regenerating enzymes it was
