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

The recombinant enzyme was modified by addition of two cysteins,arranged in front of the His-tag, to make use of the enzyme forelectroenzymatic applications. The enzyme variant exhibited unaffectedactivity in solution and the cystein-residues enabled a directedimmobilization of the enzyme to gold electrodes without a further linkermolecule.The electrochemical reoxidation of the co-substrate NAD+, hencethe successful biofunctionalization of the electrode was monitored by cyclicvoltammetry (CV) [2,3]. Determination of CVs with increasing substrateconcentrations enabled Km-value determinations of 0.06 mM, exhibiting aslightly increased affinity to the biochemical value in solution of 0.2 mM.By use of bioinformatic methods several amino acid exchanges have beensuggested for developing enzymes with increased stability and the mutantshave been constructed.[1] Schneider, K.H. et al (1995): Microbiology 141, 1865-1873.[2] Gajdzik, J. et al (2007): J. Solid State Electrochem. 11, 144-149.[3] Kornberger P. et al (2009): Langmuir 25, 12380-12386.[4] Carius, Y. et al (2010). J. Biol. Chem. 285, 20006-20014.GWP004Uptake and utilization of glucosamine inCorynebacterium glutamicumA. Uhde*, T. Maeda, V. Liedschulte, G. Seibold, R. Krämer, K. MarinInstitute of Biochemistry, University of Cologne, Cologne, GermanyCorynebacterium glutamicum is a Gram-positive biotin-auxotrophicactinobacterium primarily applied for the industrial production of aminoacids like L-glutamate and L-lysine. Since feedstock costs hold a significantpart of overall fermentation costs it is desirable to use low-cost, renewableresources like hydrolysed lignocellulose and/or chitin-rich polysaccharides.Such feedstocks contain a variety of different carbohydrates includingglucose, glucosamine or N-acetylglucosamine. Interestingly, utilization ofamino sugars was not investigated for C. glutamicum yet. We addresseduptake of glucosamine to explore the metabolic set-up for its utilization.C. glutamicum is able to grow on glucosamine as sole carbon and nitrogensource, but only with half the growth rate than on glucose as carbon sourcewas observed. In contrast to many other substrates glucosamine and glucoseare not simultaneously metabolized by C. glutamicum. Since a hpr mutant ofC. glutamicum is not capable to utilize glucosamine participation of a PTStypecarrier (phosphostransferase systems) was indicated. Growth assayswith mutants lacking PtsG (glucose), PtsF (fructose) or PtsS (sucrose)revealed that PtsG is the sole uptake system for glucosamine in C.glutamicum. Transport assays with radiolabeled substrates were performedto characterize the kinetics of glucoseamine uptake indicating that PtsGmediates glucoamine uptake as side activity. The impact of the PtsGdependent glucosamine uptake on the efficiency of its utilization will bediscussed.which mixed fermentation kinetics were observed. Bacteriocin productioncommenced only when the broth pH dropped to levels of ~3.5 due to acidproduction at approximately 15 hours.These observations suggest that an oxidative metabolic pathway is involvedin biosynthesis and that conversion of prepeptides to active peptides takesplace upon acidification of the broth. Irrespective of the microorganismused, a feeding strategy for maintaining the pH at 3.5 and glucose at 20 g/Lfrom 10 hours, extended the production period and almost doubled the yieldof secreted proteins in all cases.GWP006Pediocin production from Weissella paramesenteroides infermentation systems: Glucose feeding strategies andyields.M. Papagianni*School of Veterinary Medicine, Food Hygiene and Technology,Thessaloniki, GreeceAn atypical meat isolated Weissella paramesenteroides strain was found toproduce a class II a bacteriocin of approximately 5 kDa M.W. Thebacteriocin was partly characterized and studied with respect of variousphysicochemical and biochemical properties of its molecule. Following thebacteriocin molecule studies, attempts were made to optimize production infermentations carried out in a 3-L stirred tank bioreactor.The effect of the concentration of glucose in the substrate was studied inbatch cultures in which initial concentrations varied between 10 and 30 g/L.The glucose effect was also studied in fed-batch cultures operated asglucostats and pH-stats. In these runs feeding started at 10 hours whileglucose and pH were maintained at stable levels: 10, 15, 20, 25, and 30 g/Lglucose and pH 3.5. Another feeding strategy was also applied in which aglucose solution was fed in single shots in batch runs with initial glucoseconcentration of 10, 15, and 20 g/L. Maximum bacteriocin production levelsin batch cultures reached 850 AU/ml under semiaerobic conditions and with25 g/L initial glucose concentration.Maintaining stable levels of glucose and pH extended the production periodin all runs however, an optimum was observed for glucose concentrationlevels at 25 g/L, at which production levels were the highest (1500 AU/ml).Fermentation process kinetic studies showed growth-associated productionfor glucose levels ≥20 g/L. Substituting the production medium with glucoseat time-points at which specific production rates started to drop resulted inhigher specific productivities however, the optimum concentration of 25 g/Lglucose was again observed in this type of feeding.Therefore, the concentration of the carbon source plays an important role inbacteriocin production from W. paramesenteroides and manipulating itslevels by choosing the appropriate mode of fermentation can lead tosignificant increases in process productivities.GWP005Class IIa bacteriocins from Pediococci: Production infermentation systemsM. Papagianni*School of Veterinary Medicine, Food Hygiene and Technology,Thessaloniki, GreeceClass IIa bacteriocins of lactic acid bacteria are structurally related proteinswith a molecular weight ranging between 2-6 kDa. Their N-terminuscontains a highly conserved region known as the pediocin box, with thecharacteristic amino acid sequence of -YGNGV-. These bacteriocins areknown as the pediocin-like and Listeria-active bacteriocins. Since the firstisolated pediocin PA-1 from Pediococcus acidilactici, a number of otherpediocins from more or less related species have been isolated and partlycharacterized. These bacteriocins, apart from their broad inhibition spectratowards Gram-positive food spoilage and pathogenic bacteria, exhibittechnologically important properties -e.g. maintenance of activity atsterilization or freezing temperatures and at pHs ranging from 2 to 10, whichmake them important potential biopreservatives.In this work, we report on the common characteristics of production of thenewly identified pediocins from P. acidilactici, P. pentosaceus and P.damnosus in batch and fed-batch fermentation systems. Kinetic studies onfermentations carried out under different dissolved oxygen levels in a 3-Lstirred tank bioreactor, run at 150 rpm and 30 o C, revealed that in all casesDO levels of 50-60% supported bacteriocin production with growthassociatedand primary metabolite kinetic patterns and higher specificproductivities compared to anaerobic or fully aerobic conditions underGWP007Characterization of the 1,3-PD dehydrogenase ofClostridium sp. IBUN 13A and establishment of anenzyme assay for measuring 1,3-PDS. Franz 1 , J. Montoya* 1 , D. Montoya 2 , P. Dürre 1 , B. Schiel-Bengelsdorf 11 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,Germany2 Institute of Biotechnology, National University of Colombia , Bogota D.C.,ColombiaThe non-pathogen strains Clostridium sp. IBUN 158B and IBUN 13A wereisolated from the soil of Colombian tomato and potato fields. These strainsbelong to a new species, closely related to C. butyricum, and are able totransform residual glycerol from biodiesel production into 1,3-propanediol(1,3-PD). In order to establish a competitive industry based on these strains,their 1,3-PD yield should be increased by means of genetic engineering. Formeasurement of 1,3-PD, an enzymatic assay based on the enzyme 1,3-PDdehydrogenase of strain IBUN 13A (encoded by the dhaT gene) wasestablished. This NAD + -dependent enzyme converts 1,3-PD into 3-hydroxypropionaldehyde, and the proportional amount of NADH producedcan be detected spectrophotometrically at 340 nm. In order to overproducethe enzyme, the dhaT gene of IBUN 13A was cloned into the vector pET-28a(+) of Novagen® (which adds an N-terminal his-tag to the enzyme), andthe resulting plasmid was transformed into E. coli BL21(DE3). As theenzyme is sensitive to oxygen, the whole purification process was performedunder anaerobic conditions. After cell disruption using a French Press, thespektrum | Tagungsband 2011