VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

EPS matrix showed that it consists mainly of polysaccharides (50%),proteinaceous compounds (20%), and lipids (10%), while humic acids,uronic acids, and nucleic acids account for the rest of the matrix. Styrenestimulated substantially the production polysaccharides compared to nonsolventconditions. This study underlines the enhanced robustness ofbiofilms in solvent saturated environments, which is supposedly acombination of effects such as the increased EPS production, physiologicalchanges due to adaptation, slow growth and other resistance mechanisms.[1] Halan, B. et al (2010): Maximizing the productivity of catalytic biofilms on solid supports inmembrane aerated reactors. Biotechnol Bioeng: 106: 516-527.[2] Halan, B. et al (2010): Real time solvent tolerance analysis of Pseudomonas sp. strain VLB120ΔCcatalytic biofilms (submitted).GWV020Asymmetric benzylic hydroxylation and epoxidation ofalkylbenzenes and styrene derivatives by Agrocybeaegerita aromatic peroxygenaseM. Kluge* 1 , R. Ullrich 1 , K. Scheibner 2 , M. Hofrichter 11 Bio- and Environmental Sciences, International Graduate School (IHI)Zittau, Zittau, Germany2 Biology, Chemistry and Process Technology, University of AppliedSciences Lausitz, Senftenberg, GermanyOptically pure C α-hydroxy alkylbenzenes and C α-C β styrene epoxides are ofvital interest as building blocks in the synthesis of pharmaceuticals and finechemicals. In particular chiral epoxides offer two defined stereo centers inring opening reactions. The agaric mushroom Agrocybe aegerita produces aheme-thiolate peroxygenase (AaeAPO) belonging to a new class of aromaticperoxygenases (APOs) that shares spectral and catalytic properties both withperoxidases and cytochrome P450 monooxygenases and catalyses variousoxygenation reactions. Hydroxylations and epoxidations proceed withsometimes virtually perfect stereoselectivity yielding phenyl alcohols andoxiranes, respectively. Typical substrates are non activated hydrocarbonssuch as alkylbenzenes and styrene derivatives as well as their cycloalkylbenzene analogons. Enantiomeric excesses greater than 99% and totalturnovers up to 110,000 cycles are achieved. Besides this, the apparentkinetic K M- (k cat-) values determined for ethyl and propyl benzenehydroxylation to 694 μM (409 s -1 ) and 480 μM (194 s -1 ) supportimplementation into technical processes. Mechanistic aspects regardingstereoselectivity and oxygen incorporation are further objects of discussion.GWP001Characterization of immobilized alkaline cyclodextringlycosyltransferase from a newly isolated Bacillusagaradhaerens KSU-A11A. Ibrahim*, M. El-Tayeb, A. Al-SalamahFaculty of Science, Department of Microbiology, King Saud University,Riyadh, Saudi ArabiaAlkaliphilic bacteria were isolated from soil and water samples obtainedfrom Egyptian soda lakes (Wadi Natrun area, Egypt). Screening forcyclodextrin glycosyltransferase-producing alkaliphilic bacteria resulted inisolation of 10 positive strains. Strain KSU-A11 was selected as the bestCGTase producer (2.1 U/ml). 16S rDNA sequence analysis identified theKSU-A11strain as Bacillus agaradhaerens. CGTase was partially purifiedusing starch adsorption technique. The partially purified CGTase wasimmobilized on chitin by covalent binding tecnique using cross linkingreaction with high immobilization yield (85%). The properties of the freeand immobilized CGTase were determined. The optimum pH of theimmobilized enzyme was slightly higher than that of the free enzyme, pH 10and 10.5 respectively. In addition, both free and immobilized CGTaseretained 94 to 100 % of its initial activity over a wide pH range (pH 6.0 to11.0). The enzymatic activity of both free and immobilized CGTase washighest at temperature 50 ºC; however, the relative activities of theimmobilized enzyme were slightly higher than those of the free enzyme.Furthermore, investigation of thermostability of the enzyme indicated thatthe immobilization process of CGTase on chitin significantly protected theenzyme against thermo-inactivation. Kinetic parameters Km and Vmaxvalues for free and immobilized enzymes were estimated and while therewas no change in the Vmax value for both free and immobilized CGTase(83.3 μmol/min. mg), the Km of the enzyme increased from 14.28 to 20mg/ml upon immobilization. The immobilization of the enzyme showedhigh operational stability by retaining about 50% of the initial activity afternine uses.GWP002Investigations of microalgal growth kinetics and theflashing light effectA. Jacobi*, C. PostenInstitute of Engineering in Life Sciences, Bioprocess Engineering, KarlsruheInstitute of Technology (KIT), Karlsruhe, GermanyThe construction of economic pilot plant photobioreactors exhibits stillmajor problems due to insufficient scale-up of technical parameters from labto larger scale. The layout of large scale photobioreactors requires theknowledge of the physiological reactions of the specific strain(s) moreoverthe resulting kinetics of growth and product formation under differentillumination conditions (light intensity, frequency of light/dark cycles) are offundamental interest and importance. Therefore a scale-down approach wassuccessfully applied for identification and determination of these relevantand critic parameters. The conditions in one volume element of a largereactor are mimicked in a small scale model reactor. The configuration andsuccessful application of this model reactor for investigation ofChlamydomonas’ growth under constant and flashing light conditions willbe presented in this contribution.The model reactor comprises a special designed illumination devicedeveloped using light emitting diodes (LEDs) and collimating lensesresulting in an entirely homogenous illuminated volume. The focusing effect(lens effect) of the light source to the center of the reactor compensates themutual shading of the algae cells. Measurements of light intensitydistribution inside the reactor containing media of different optical densitieswill be shown which could verify the homogenous illumination in rangesrelevant for determination of growth kinetics.Besides the model reactor was applied for kinetic investigations of differentalgae in various operating modes (Batch, Fed-Batch and ContinuousCulture). For instance the effect of reduced antenna size on growth rates ofChlamydomonas reinhardtii mutants at different light intensities comparedto the non-mutated strain will be depicted. Experiments have beenperformed under constant illumination up to very high, saturating andalready inhibiting light conditions. Furthermore the positive effect of fastlight/dark cycles on growth rate (flashing light effect) was examined andrelevant rates were identified.[1] Rosello Sastre, R. et al (2007): Scale-down of microalgae cultivations in tubular photo-bioreactors-a conceptual approach. Journal of Biotechnology 132 (2):127-133.GWP003Modified Galactitol-Dehydrogenase from Rhodobactersphaeroides D for Electrochemical ApplicationsG.-W. Kohring* 1 , S. Gauer 1 , P. Kornberger 1 , C. Gumhold 1 , J. Gajdzik 2 ,R. Hempelmann 2 , C. Søndergaad 3 , J. Jensen 3 , H. Christian 4 , A. Faust 4 ,Y. Carius 5 , A. Scheidig 4 , F. Giffhorn 11 Institite for Microbiology, Saarland University, Saarbrücken, Germany2 Department of Physical Chemistry, Saarland University, Saarbrücken,Germany3 Bioinformatics Center, University of Copenhagen, Copenhagen, Denmark4 Structural Biology, University of Kiel, Kiel, Germany5 Department of Structural Biology, Saarland University, Homburg,GermanyDehydrogenases represent an important class of enzymes in biotechnology.By enantioselective reduction or oxidation they provide access to rare sugarsand alcohols which may serve as optically pure compounds and chiralbuilding blocks in the pharmaceutical and chemical industry.The 765 bp sequence of the galactitol dehydrogenase [1] (GatDH) genecoding for a subunit of the homotetrameric enzyme with 254 amino acidsand a molecular mass of 26.4 kDa was functionally expressed in Escherichiacoli BL21Gold(DE3). The heterologously expressed GatDH was elongatedby the attachment of a His(6)-tag to the N-terminus of the protein whichfacilitated the purification and did not affect the catalytic activity. The activeconformation is strictly dependent on the presence of bivalent cations likeMg2+. The crystal structure revealed that the Mg-ions are coordinated bythe last three amino acids of the C-termini from two dimers, which let thetetramer appear as a dimer of dimmers [4]. To illustrate the mechanisms ofthe active site, the deduced oxidation of pentanediol is depicted.spektrum | Tagungsband 2011