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ew Biotechnology · Volume 29S · September 2012

oster 5.0.135

dentification of IS2 transposition in pVAX1-based plas-id, a common vector for DNA vaccine development

.A.L. Goncalves1,2,∗, P.H. Oliveira2, L.A. Lewis3, D.M.F.razeres1,2, G.A. Monteiro1,2

MIT-Portugal Program, PortugalDepartment of Bioengineering and Institute for Biotechnology and Bio-ngineering, Instituto Superior Técnico (IST), Technical University ofisbon, Lisboa, PortugalDepartment of Biology, York College of the City University of New York,

amaica, NY 11451, USA

ontamination of plasmid DNA molecules by host insertionequences can alter their biological properties and even compro-ise safety if therapeutic applications are envisaged. Here, we

eport the detection of IS2 transposition in two different regionsf the plasmid pVAX1-GFP: upstream the neomycin resistanceene and within the GFP gene. According to our previous observa-ions [1], both regions were found to exhibit intrinsic curvature.lthough no significant differences in the number of IS2 copiesere observed during prolonged growth of Escherichia coli DH5�

earing pVAX1-based plasmids, the GFP-associated transpositionas found to occur at approximately twice the frequency than

he neomycin-associated one. We also looked at the influence ofedium composition on IS2 transposition, by culturing cells inL fed-batch bioreactors at 37◦C and 30% D.O.T. in either semi-efined medium (SDM) or complex medium (CM). Real-time PCRuantification of IS2 transposition revealed a 3-fold increase in theumber of insertions occurring in SDM than in CM, and almostxclusively within the GFP gene. It is possible that under nutri-ional stress the transposase activity is enhanced, thus favouringS2 transposition. Our results add to previous evidence that impli-ate nutrient limitation in enhanced IS transposition [2]. Also,hey raise the question of what parameters are behind the higherransposition frequency within the GFP gene. Ultimately, the iden-ification of IS2 insertions in pVAX1-GFP represents an alert forlasmid biopharmaceutical research, since this vector is frequentlysed for DNA vaccine development.

Keywords: Insertion sequence; Plasmid stability; Fermenta-ion strategy; DNA vaccines

eferences

. Lewis, et al. Mob DNA 2012;3:1.

. Twiss, et al. Mol Microbiol 2005;57:1593–607.

ttp://dx.doi.org/10.1016/j.nbt.2012.08.575

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oster 5.0.136

odeling of solid-state cultivation of Trichoderma inrder to obtain natural substances with high cationxchange capacity

ergana Angelova∗, Konstantin Chakalov, Todorka Popova,alentin Savov

ROMB Ltd, 1Aboba Str., 1606 Sofia, Bulgaria

he influence of the supporting mineral matrix (zeolite, ver-iculite or diatomite) on the formation of humic acids (HA)ith high cation exchange capacity (CEC) in the case of solid-

tate fermentation of Trichoderma on organic substrates – lignite,eonardite or peat – is examined. The combination of lignite withiatomite or vermiculite during the solid-state fermentation pro-ides 20–30% higher yields of HA than the combination witheolite. T. viride has a stronger influence on the biotransformationf lignite than T. harzianum. Vermiculite and diatomite contributeo the formation of more fulvic acids than zeolite. CEC of zeolites maintained and even partially increased in the case of biotrans-ormation of lignite with Trichoderma. Obviously, zeolite is able todsorb Cu better than vermiculite and diatomite and therefore rel-tively less Cu ions pass into the HA. A significant proportion ofu remains in the insoluble residue of leonardite. HA of leonardite

orbs 100% of the extracted Zn and 0% of Co. HA formed in thease of solid-state fermentation with lignite in combination withiatomite have the highest capacity to sorb Mn. CEC is proportion-tely reduced by the addition of vermiculite and diatomite whilehe addition of zeolite slightly increases it. The sorption propertiesf HA depend on the extent of humification of the organic matterith a significant correlation.

Keywords: HA; Trichoderma

ttp://dx.doi.org/10.1016/j.nbt.2012.08.576

oster 5.0.137

valuation of the effect of substrate amount, sulfuriccid concentration and reaction temperature on acidicydrolysis of cellulose casings applying a central com-osite design

esús J. Vidal-Vidal, Guillermo Arzate-Martínez∗, Lizzette Moreno-arcía, Oscar M. Portilla-Rivera

Universidad Politécnica de Guanajuato, Av. Universidad Norte s/n Loc,uan Alonso, Cortazar Guanajuato, Mexico

cellulose casing is a film used on the preparation of extrudedeat products; which functions as their outer cover while being

ooked. Its disposal has been a problem because of its large pro-uced volumes. Applications on its reuse have had limited use dueo its complexity; so its hydrolysis to produce glucose could be con-idered, as it can be used as a substrate for microbial applications;his can be accomplished with the use of inorganic acids. In thisork, we evaluated the effect of three process factors on the acidic

ydrolysis of these cellulose films. Shredded cellulose film wasixed with sulfuric acid on a heated caped flask. The tested factorsere: cellulose film concentration (4–10%, w/w), sulfuric acid con-

entration (5–25%, w/w) and temperature (53–87◦C). Our response

www.elsevier.com/locate/nbt S205

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Keywords: Lipase; Biosurfactant; Wastewater treatment

http://dx.doi.org/10.1016/j.nbt.2012.08.579

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ariables were: Final reducing sugars concentration, % celluloseonversion and sugar production rate during 48 h. Employing aentral composite design, we found that an increase on the tem-erature and the film and acid concentrations, the hydrolysis ratend reducing sugars concentration augmented; nevertheless, acidoncentration did not have effect for lower film concentrations.ellulose conversion increased as acid concentration and the tem-erature were raised. The best conditions for this reaction were:% film concentration, 21% of acid and 80◦C, where we obtainededucing sugar concentration of 3.56%, conversion of 60.85% andugar production of 0.1944 g sugar/(h*250 g reaction medium). Allurface response graphs were linear; so, further experimental workhould be done to find the optimal hydrolysis conditions.

ttp://dx.doi.org/10.1016/j.nbt.2012.08.577

oster 5.0.138

ecretory production of virus-like particles by recombi-ant insect cells

ideki Yamaji1,∗, Miwa Kuwahara2,3, Eiji Konishi2,3,4

Department of Chemical Science and Engineering, Graduate Schoolf Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501,apanDepartment of International Health, Kobe University Graduate Schoolf Health Sciences, 7-10-2 Tomogaoka, Suma, Kobe 654-0142, JapanBIKEN Endowed Department of Dengue Vaccine Development, Fac-lty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road,atchathewi, Bangkok 10400, ThailandDivision of Vaccinology, Center for Infectious Diseases, Kobe Universityraduate School of Medicine, 7-5-1 Kusunoki, Chuo, Kobe, Japan

irus-like particles (VLPs) have been developed as candidate vac-ines because they are safe and structurally similar to authenticiruses. In this study, we investigated the production of Japanesencephalitis (JE) VLPs in stably transformed lepidopteran insectells. The DNA fragment encoding the JE virus prM signal pep-ide, the precursor (prM) of the viral membrane protein (M), andhe envelope glycoprotein (E) was cloned into the plasmid vectorIHAbla. The pIHAbla contained the Bombyx mori actin promoterownstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-transactivator and the BmNPV HR3 enhancer for high-level

xpression, together with a blasticidin resistance gene for use asselectable marker. A DNA encoding a form of prM with a pr/M

leavage site mutation was used to suppress cell-fusion activity ofLPs. After transfection with the resulting plasmid, Trichoplusia niTI-TN-5B1-4 (High Five) cells were incubated with blastcidin, andells resistant to the antibiotics were obtained. Western blot anal-sis and enzyme-linked immunosorbent assay (ELISA) of a cultureupernatant showed that transfected High Five cells secreted the Entigen equivalent to the authentic JEV E. Sucrose density-gradientedimentation analysis of the culture supernatant suggested thatecreted E antigen molecules were produced in a particulate form.LPs recovered from the supernatant were able to induce neutral-

zing antibodies in mice, especially when emulsified with alum

djuvant. High yields of E antigen of more than 10 mg/L waschieved in serum-free suspension cultures. These results indicate

206 www.elsevier.com/locate/nbt

New Biotechnology · Volume 29S · September 2012

hat recombinant insect cells may offer a novel approach for effi-ient VLP production.

ttp://dx.doi.org/10.1016/j.nbt.2012.08.578

oster 5.0.139

ipase and biosurfactant application on the pre-ydrolysis and anaerobic biologic treatment of effluentsith high fat contents

.N. Silva2, M.L.E. Gutarra2,∗, D.M.G. Freire1, M.C. Cammarota2

Universidade Federal do Rio de Janeiro, Instituto de Química Rio deaneiro, RJ, BrazilUniversidade Federal do Rio de Janeiro, Escola de Química Rio de

aneiro, RJ, Brazil

ipases can be applied in a large variety of biotechnological appli-ation including treatment of effluents with high fat contents.iologic anaerobic treatment of this kind of effluent has someroblems caused by high fat contents. Thus, the pre-treatmentith enzymes has been proposed as an alternative or a com-lement to biological conventional process. Other alternative ishe use of biosurfactants that can make fats and oils more avail-ble for microbial biomass. In this way, to promote an efficientreatment of effluents with high fat contents, we evaluated theombined action of a liquid enzymatic preparation from Penicil-ium simplicissimum obtained by solid state fermentation and aammnolipid biosurfactant produced by Pseudomonas aeruginosan pre-hydrolysis followed by a biologic anaerobic treatment ofn effluent from a poultry slaughterhouse. Highest free fat acidoncentration (14 �mol/mL) was obtained with 6.2% (v/v) of liq-id enzymatic preparation (lipase activity 4.7 U/mL), 0.4% (w/v)iosurfactant (from a stock solution of 4.9 g/L ramnolipid) and4◦C. For anaerobic treatment was used the effluent pre-treatedn two different conditions, with high concentration of freeat acids (14 �mol/mL) and other with medium concentration8 �mol/mL). Similar DQO removal were found in both con-ition but higher methane production (161 mL of CH4/g DQO)ere obtained with the effluent with lower free fat acid contentbtained after pre-hydrolysis step (8 �mol/mL), while with theffluent that was not submitted to the hydrolysis step no methaneas produced probably because of the high contents of oil and