S398 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

References

Beccari, M., et al., 2009. J Chem Technol Biotechnol 84, 901–908.Dionisi, D., et al., 2005. Wat Res 39, 2076–2084.

doi:10.1016/j.jbiotec.2010.09.515

[P-I.150]

Evaluation of the Recombinant E. coli BL21 Strains ProducingProchymosin in the Biotechnological Processes

Leila Badiefar ∗, Mahvash Khodabande, Gholamreza Ahmadian

National Institute of Genetic Engineering and Biotechnology, IslamicRepublic of IranKeywords: Prochymosin; Protein expression; Sulobilization;Refolding

The levels of protein expression and the amount of cell bulkare some of the very important parameters in the biotechnologicalprocesses involving high-yield production. Chymosin is an aspartylproteinase that is found in the abomasums of unweaned calves.In this research, we compared protein expression levels, total cellmass and production efficiency at two recombinant E. coli BL21 car-rying the complete and exon-6 less prochymosin under flask andfermenter culture conditions. Studied variables were IPTG concen-tration, OD for induction of protein production, time after inductionand temperature. The best growth conditions was selected in therange of variables under experiment. The level of prochymosin pro-duction and the final OD were measured at mentioned conditions.Also, the production efficiencies of the two prochymosin formswere evaluated. The level of prochymosin production was thencompared by fermentation of two recombinant E. coli BL21 strainsunder the optimized conditions. The results indicate that the levelsof protein expression and final OD under optimum conditions are33.53 and 5.88 for E. coli BL21 harboring pET- prochymosin and 45.4and 7.82 for E. coli BL21 harboring pET- exon 6less prochymosin,thus their production efficincies will be 197.15 and 355.02 respec-tively. Whereas every two types of prochymosin are producedas insuloble inclusion bodies in the cytoplasmic space, sulobi-lization and refolding of these proteins are necessary. We couldsolubilize and refold complete and shortened prochymosin withefficiency of 47 and 56% respectively. Therefore the results relatedto the efficiencies of the prochymosin production, solubilizationand refolding of inclusion bodies indicate that use of E. coli BL21harboring pET- exon 6less prochymosin is very better than E. coliBL21 harboring pET- prochymosin at biotechnological process.

doi:10.1016/j.jbiotec.2010.09.516

[P-I.151]

Separation of p-aminobenzoic acid using liquid membrane inpresence of phase modifier

L. Kloetzer 1, A.C. Blaga 1,∗, A.I. Galaction 2, D. Cascaval 1

1 Technical University of Iasi, Romania2 University of Medicine and Pharmacy of Iasi, RomaniaKeywords: p-aminobenzoic acid; Pertraction; Liquid membrane;Phase modifier

The studies on facilitated pertraction with Amberlite LA-2 ofPABA in presence of 1-octanol added into the membrane phaseindicated the significant increase of the acid initial and final massflows. Compared with the facilitated pertraction without alcohol,

Fig. 1. Influence of pHS on factors FN and FP (pHF = 4, Amberlite LA-2 concentra-tion = 40 g/l, rotation speed = 500 rpm; 1-octanol concentration = 10% vol.).

the acid mass flows have been amplified up to 3 times, but themagnitude of this effect has to be correlated with the pH-gradientbetween the feed and stripping phase, carrier and 1-octanol con-centrations inside the liquid membrane and mixing intensity. Fig. 1.

Contrary to this positive effect, the addition of 1-octanol inducedthe diminution of the permeability factor, because the extractionrate of PABA into the membrane phase exceeded that of reextrac-tion from this phase, the transport capacity of the investigatedpertraction system being limited. For this reason, the aim of thefuture studies is to optimize the reextraction process of the acidfrom the liquid membrane to the stripping phase.

doi:10.1016/j.jbiotec.2010.09.517

[P-I.152]

Bioprocess systems engineering applied to a multipurpose sec-ond generation ethanol-from-sugarcane plant: assessing theperformance of local and global optimization algorithms

F.F. Furlan 1,4, R.C. Giordano 1,4,∗, A.J.G. Cruz 1,4, C.B.B. Costa 1,4,A.R. Secchi 2,4, R.P. Soares 3,4

1 Federal University of Sao Carlos, Brazil2 COPPE, Federal University of Rio de Janeiro, Brazil3 Federal University of Rio Grande do Sul, Brazil4 Chemtech, BrazilKeywords: Biorefinery optimization; Second generationbioethanol; Sugarcane bagasse enzymatic hydrolysis; BioprocessSystems Engineering

Production of biofuels is presently at a technological crossroad. Ahard competition among technologies is in course and the winnerswill be defined by a combination of economical criteria, complianceto environmental restrictions, and process robustness.

Optimization of this complex and integrated industrial processmust aim at different targets: cost reduction, sustainability (use ofland, negative CO2 balance), effluent emissions, and water usagecutback. In this scenario, fine-tuned processes, operating at (near-)optimum conditions will have significant competitive advantage,and the application of Bioprocess Systems Engineering tools will beessential for this task.

This work assesses the performance of two algorithmsfor defining optimal operation points of a second generationbioethanol-from-sugarcane plant. A highly energy-integrated pro-cess, using surplus bagasse and lignin for steam generation, is usedas case study. First and second generation anhydrous and hydrated

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S399

ethanol, electric power and bagasse for animal feeding are productsof this multipurpose biorefinery, employing enzymatic hydroly-sis of cellulose and hemicellulose. For a first approach, a classicaleconomical cost function is defined.

This is a non-linear optimization problem with constraints,solved through two algorithms: Sequential Quadratic Program-ming, a local search method, and Particle Swarm Optimiza-tion, a global method including the constraints as penaltyfunctions. The process is simulated using the applicative Envi-ronment for Modeling, Simulation, and Optimization (EMSO,www.enq.ufrgs.br/trac/alsoc/wiki/EMSO). Our results show thatthe capability of the global optimization method to avoid localminima may be important, despite being more computationalintensive, to find the most competitive technology.

doi:10.1016/j.jbiotec.2010.09.518

[P-I.153]

Synthesis of Galacto-oligosaccharides in Repeated Batch Opera-tion with Immobilized b-galactosidase from Aspergillus oryzae

A. Illanes ∗, L.M. Huerta, C. Guerrero, C. Vera

Pontificia Universidad Católica de Valparaíso, ChileKeywords: Galacto-oligosaccharides; Prebiotic; Immobilizedenzyme; Enzyme reactor

Introduction: Lactose-derived prebiotics may be obtained fromwhey that contains most of the lactose in milk. Lactose conver-sion into functional food ingredients, represents a significant addedvalue; several of such products have been evaluated, galacto-oligosaccharides (GOS) outstanding among them. Here we presentresults on the synthesis of GOS at very high lactose concentra-tions with immobilized �-galactosidases from Aspergillus oryzae,highlighting the potential of using immobilized biocatalysts insequential batch reactor operation in terms of specific productivityand biocatalyst efficiency

Methods: �-galactosidase from Aspergillus oryzae (Enzeco fungallactase) was immobilized by multi-point covalent attachment toglyoxyl agarose (�-GAL-GA). The synthesis of GOS was conductedat pH 4.5 and 50 ◦C and optimized with respect to lactose concen-tration and enzyme to substrate ratio using lactose conversion intoGOS (XGOS) as objective function. Sequential batch reactor opera-tion was carried out at the optimum conditions, each batch beingconducted up to the point of maximum conversion; after each batchthe biocatalyst was recovered and used without make-up in thefollowing batch

Results and Discussion: Optimum conditions for GOS synthe-sis were 54.6% (w/v) lactose concentration and 33 internationalunits of �-galactosidase /g lactose, obtaining an XGOS of 28.7%.Reactor operation was conducted for ten sequential batches andat such conditions the enzyme was quite stable with a half-lifevalue of 260 h, 53% higher than under non reactive conditions. XGOSremained constant around 29% during the operation cycle and atthe end of which 8500 g of GOS per gram of enzyme preparationwere produced, with a biocatalyst efficiency increase of 400% withrespect to the free enzyme in solution. This amount can be fur-ther increased by considering additional batches up to the point ofbiocatalyst replacement

Acknowledgements

Work funded by Chilean Fondecyt Grant 1100050.

doi:10.1016/j.jbiotec.2010.09.519

[P-I.154]

Optimization of soluble expression of recombinant ther-mophilic nucleoside phosphorylases

K. Szeker 1,∗, M. Casteleijn 2, P. Neubauer 1

1 TU Berlin, Institut für Biotechnologie, Germany2 University of Oulu, Bioprocess Engineering Laboratory, FinlandKeywords: Protein expression; Recombinant; Nucleoside phospho-rylase

Nucleoside Phosphorylases are enzymes of particular interestfor the biocatalytic preparation of nucleoside analogues, which dis-play an important class of pharmaceutical agents and are usedfor molecular biological techniques and diagnostics. However, thebiocatalytic process is often limited by i) the instability of the bio-catalyst, in particular at elevated temperatures, and ii) a lack ofactivity towards modified nucleoside substrates.

We investigate the recombinant expression of Nucleoside Phos-phorylases isolated from thermophilic microorganisms in E. coli.Our approach includes the stabilization of an expression vectorby introducing the parB locus of the plasmid R1, leading to post-segregational killing of plasmid-free cells, and the coexpressionof chaperones. Using Purine Nucleoside Phosphorylases, isolatedfrom Deinococcus geothermalis, we show that this constitutes asuccessful strategy to significantly increase the soluble fraction ofthe expressed protein. Furthermore, we discuss how to optimizethe soluble protein expression by means of experimental designand high cell density cultivations in small-scale.

doi:10.1016/j.jbiotec.2010.09.520

[P-I.155]

Selection of natural material for Saccharomyces cerevisiaeimmobilization

Z. Genisheva ∗, S.I. Mussatto, J.M. Oliveira, J.A. Teixeira

Institute for Biotechnology and Bioengineering, University of Minho,PortugalKeywords: Immobilization; Yeast; Natural

Immobilized cells systems have been considered as a promis-ing alternative to improve the performance of biotechnologicalprocesses, since in these systems, immobilized cells completelymaintain their biological functions with increased stability thatmay often lead to increased cell productivity. However, the correctselection of immobilization carrier is essential to design an effec-tive system to each particular purpose. The objective of the presentstudy was to find a cheap and abundant natural material suitable forimmobilization of Saccharomyces cerevisiae yeast, which is usuallyused in fermentation processes for wine production. Four differ-ent carriers were evaluated, namely grape seeds, grape skin, grapestems and corn cobs.

To be used in the experiments, the carriers were washed withwater and dried to constant weight. The inoculum was preparedby cultivating the yeast in YPD medium under static conditionsfor 24 h and 30 ◦C. Fermentation runs were performed in semi-defined medium composed by: glucose (120 g/L), yeast extract(4 g/L), (NH4)2SO4 (1 g/L), KH2PO4 (1 g/L), and MgSO4 (5 g/L). Theassays were carried out in 500 mL Erlenmeyer flasks containing200 mL of medium and 2 g of material carrier, statically incubatedat 30 ◦C for 24 h. Samples were taken periodically for estimation ofbiomass, glucose consumption and ethanol production.

Corn cobs and grape skins were the best material carriers forS. cerevisiae immobilization, since they immobilized the highest