S452 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

larvae of the fleshly Neobellieria bullata were chosen as a source ofthese peptides.

The haemolymph was picked up from larvae, gradually cen-trifuged and precipitated by acidified methanol. Subsequentlythese fractions were separated by chromatographic methods (SPEcolumn, FPLC, RP-HPLC) to gain fractions of short peptides. Identifi-cation and characterization of these fractions were made by tricineelectrophoresis, mass spectrometry analysis (MS) and N-terminalsequencing.

Antimicrobial activity of fractions was screening by diffu-sion method. Tested organisms were gram-negative bacteria:Escherichia coli, Pseudomonas aeruginosa, gram-positive bacteria:Staphylococcus aureus, Bacillus megaterium, Listeria innocua, andfungi: Candida scotii, Aspergillus ochraceus and Mucor species.

Several fractions showed antimicrobial activity against bacte-ria Escherichia coli, Staphyllococcus aureus, Pseudomonas aeruginosa,Bacillus megaterium and fungi Candida scotii. Tricine electrophore-sis proved presence of lowmolecular peptide. MS analysisdetermined molecular masses less than 10 kDa. N-terminal seque-nation was successful just on partial sequence and the sequenceswe got do not have any similarity match with the database peptidewith antimicrobial properties.

The authors are thankful to the grants MSM 604/613/7305,Z 405/505/06, GACR 305/09/H008 and CACR 522/09/1693.

doi:10.1016/j.jbiotec.2010.09.657

[P-M.70]

Production of Evolved Variants of D-Amino Acid Oxidase for ACancer Enzyme Therapy

Rosini Elena ∗, Molla Gianluca, Sacchi Silvia, Pollegioni Loredano

Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “TheProtein Factory”, Politecnico di Milano and Università degli Studidell’Insubria, Varese, ItalyKeywords: Flavoprotein; Oxygen; Directed evolution; Cancer ther-apy

D-amino acid oxidase (EC 1.4.3.3, DAAO) is a FAD-containingenzyme that has recently raised interest as a biocatalyst for avariety of applications. This flavoenzyme catalyses the oxidationof D-amino acids into the corresponding �-keto acids, ammoniaand hydrogen peroxide. We previously proposed yeast DAAO in aenzyme-activating prodrug therapy of solid tumors, in which thein situ production of H2O2 by the flavoenzyme can be regulated byadministration of D-amino acids (Stegman et al., 1998). Unlikely,the in vivo application of DAAO was limited by the low local oxy-gen concentration and the high Km for this substrate (in the mMrange).

In order to optimise DAAO for this application, we nowattempted a directed evolution approach. Libraries of DAAOmutants were generated by error-prone PCR and the improvedenzyme variants were identified by means of a screening proce-dure carried out at a low oxygen concentration (2.5% saturation).The clone 305, which encodes for an enzyme variant containing fivepoint substitutions, was identified because it exhibited a high activ-ity in the presence of low oxygen and D-alanine concentrations.A detailed kinetic characterization showed that this enzyme vari-ant possesses a 10-fold decrease in Km for dioxygen as comparedto wild-type DAAO: the higher activity at low O2 and D-alanineconcentrations appears to result from a combination of minor mod-ifications of specific kinetic steps, each being of small magnitude.

Interestingly, we also demonstrated a remarkable cytotoxicactivity of DAAO plus D-alanine on mouse tumor cells, especially

when the 305 mutant enzyme was employed (Rosini et al., 2009).The evolved DAAO variant is expected to lead to a suitable tool fora cancer treatment that exploits the production of H2O2.

References

Stegman, L.D., et al., 1998. Hum. Gene Ther 9, 185–193.Rosini, E., et al., 2009. FEBS J 276, 4921–4932.

doi:10.1016/j.jbiotec.2010.09.658

[P-M.71]

Characterization and Reduction of Monoclonal Antibody Aggre-gates during Ultrafiltration Process

Zhen Lu 1,∗, Lee Allen 2, Robert Field 1, Zhanfeng Cui 1

1 Department of Engineering Science, University of Oxford, Parks Road,Oxford OX1 3PJ, UK, United Kingdom2 Lonza Biologics plc228 Bath Road, Slough, SL1 4DX, UK, United King-domKeywords: Ultrafiltration; Monoclonal antibody; Aggregation;Characterization

In a biopharmaceutical downstream process, aggregate forma-tion is common but an undesirable occurrence, which is oftenrelated to activity loss, immune reaction and other side effects. Tofacility the development of aggregate-free monoclonal antibodies(MAbs) formulations, there is an urgent need for sensitive detec-tion and characterization of aggregates, especially when monomersare excessively present (>98%). Ultrafiltration is one of industrystandards in handling high-titer fermentation and has potential topurify therapeutic proteins in manufacturing scale. A combinationof separation and analytical techniques is presented in this work.SEC-HPLC, SDS-PAGE, FT-IR and dynamic light scattering wereapplied to characterize a wide range of size of soluble and insolubleaggregates, with focus on protein-protein and protein-membraneinteractions. Based on above reliable analytical protocol, ultra-filtation membrane technology has proven an effective tool foraggregates reduction, which brings down the aggregates level from2% to 0.5% by YM100 regenerated cellulose membranes.

doi:10.1016/j.jbiotec.2010.09.659

[P-M.72]

Fatty acid evaluation in a naturally isolated strain of Chlorellavulgaris

Mohammad Ali Mobasher ∗, Mohammad Hossein Morowvat, SaraRasoul-Amini, Younes Ghasemi

Department of Pharmaceutical Biotechnology, Faculty of Pharmacyand Pharmaceutical Sciences Research Center, Shiraz University ofMedical Sciences, Islamic Republic of IranKeywords: Microalgae; Chlorella vulgaris; Fatty acids; SCP

Introduction: Nutritional supplements produced from microal-gae have been the primary focus of microalgal biotechnology formany years. Dried biomass or cell extracts produced from Chlorella,Dunaliella and Spirulina have dominated the commercial opportu-nities. They are a major source of essential long-chain and polyunsaturated fatty acids. The aim of this study is to identify the Fattyacids profile of the locally isolated Chlorella vulgaris.

Methods: The Chlorella vulgaris was isolated during a screen-ing program from soil samples collected from paddy-fields of Fars

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S453

province. Total lipids from freshly freeze dried cultures of Chlorellavulgaris was extracted and converted into fatty acid methyl estersand used for determination of different types of fatty acids by gaschromatography, mass spectrometry (GC/MS) method.

Results: The composition of fatty acids in the Chlorella vulgariswas mainly, myristic acid, palmitic acid, oleic acid, Stearic acid,Linoleic acid and Lauric acid.

Discussion: Microalgae may contain significant quantities of fatsand oils (lipids) with compositions similar to those of vegetableoils. Under certain conditions, microalgae have been reported tocontain up to 85% of the dry weight as lipids. The results showedthe presence of at least 20 fatty acids in the Chlorella vulgaris.

doi:10.1016/j.jbiotec.2010.09.660

[P-M.73]

Isolation and Antimicrobial Activity of Lectin from Schinus tere-binthifolius Leaves

F.S. Gomes, T.F. Procópio, T.A. Lima, T.H. Napoleão, L.C.B.B. Coelho,P.M.G. Paiva ∗

Universidade Federal de Pernambuco, BrazilKeywords: Brazilian pepper tree; Leaf lectin; Schinus terebinthi-folius; Antimicrobial activity

Schinus terebinthifolius (Brazilian pepper tree, Anacardiaceae)leaves have demonstrated analgesic, antirheumatic and healingeffects and are used in popular medicine to treat gingivitis, can-didiasis, mycoses and infections caused by Escherichia coli. Lectinsare proteins that interact with carbohydrates and promote ery-throcyte agglutination. The aim of this work was to isolate S.terebinthifolius leaf lectin (SteLL) and to evaluate its effect as a bio-material against Candida albicans and E. coli. The extract (10%, w/v)of leaves was prepared in 0.15 M NaCl (16 h at 4 ◦C) and treatedwith ammonium sulphate in different saturations (0-20%, 20-40%,40-60% and 60-80%). Hemagglutinating activity (HA) of fractionswas evaluated with rabbit erythrocytes and HA inhibitory assaywas performed with carbohydrates (stachiose, galactose, glucose,N-acetyl-glucosamine) and glycoproteins (azocasein, ovalbumin).The more active 40-60% fraction (specific HA: 399) was chro-matographed on chitin column equilibrated with 0.15 M NaCl andthe HA (SteLL) eluted with 1.0 M acetic acid was evaluated by poly-acrylamide gel (15%, w/v) electrophoresis (PAGE) for native acidicor basic proteins. Minimal inhibitory (MIC), bactericide (MBC)and fungicide (MFC) concentrations of SteLL were determined.All HA chromatographed was recovered in SteLL (specific HA:18,618, 4.6 mg of protein; purification fold: 46.6); SteLL activity wasreduced with galactose, glucose, N-acetyl-glucosamine, stachioseand ovalbulmin for 4,654, 4,654, 4,654, 2,327 and 581, respectively.Azocasein abolished SteLL HA. A single protein band was detectedin PAGE for basic proteins. SteLL showed antibacterial effect onE. coli (MIC and MBC of 26 and 90 �g/mL, respectively) and anti-fungal activity on C. albicans (MIC and MFC of 6.5 and 26 �g/mL,respectively). In conclusion, SteLL, a cationic chitin-binding lectinpurified in milligram quantities by one chromatographic step, is abiomaterial of potential antimicrobial application.

doi:10.1016/j.jbiotec.2010.09.661

[P-M.74]

Deleterious effects of Microgramma vaccinifolia Rhizome Lectinon Fusarium species and Artemia salina

L.P. Albuquerque 1, G.M.S. Santana 1, A.M.A. Melo 2, L.C.B.B.Coelho 1, M.V. Silva 1, P.M.G. Paiva 1,∗

1 Departamento de Bioquimica, Universidade Federal de Pernambuco,Brazil2 Departamento de Biofísica e Radiobiologia, Universidade Federal dePernambuco, BrazilKeywords: Microgramma vaccinifolia; Lectin; Fusarium oxysporumf.sp. lycopersici; Artemia salina

Rhizome of Microgramma vaccinifolia is used in popularmedicine for the treatment of diarrhea and respiratory tract infec-tions. The ability of lectins to bind carbohydrates expressed ondifferent cell surfaces results in biological properties. The aims ofthis study were to isolated, characterize and evaluate antimicrobialand toxic activities of M. vaccinifolia rhizome lectin (MvRL). Rhi-zome proteins extracted with 0.15 M NaCl and precipitated with60% (w/v) ammonium sulphate were chromatographed (6.0 mg ofprotein) on Sephadex G-25 column; MvRL was collected in thewashing chromatographic step. The lectin agglutinated human ery-throcytes was thermo-stable and active at pH 5.0. Carbohydratespecificity was defined by hemagglutinating activity inhibitionassay and MvRL was a mannose-specific lectin; hemagglutinat-ing activity was also inhibited by glycoproteins from races 1, 2and 3 of Fusarium oxysporum f. sp. Lycopersici (fungus that causetomato vascular disease). MvRL was an acidic protein and SDS-PAGE revealed glycosylated polypeptide of molecular mass 17 kDa.MvRL showed a 100 kDa protein by gel filtration on Sephacryl S-300. Antifungal activity of MvRL was detected on F. decemcellulare,F. lateritium, F. moniliforme, F. solani and F. oxysporum f.sp. lycopersiciraces 1, 2 and 3; inhibition of race 3 growth (61%) was higher thanraces 1 (55%) and 2 (45%). The lectin did not show antibacterialactivity on Gram-positive (Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae,Streptococcus pyogenes) and Gram-negative (Escherichia coli, Kleb-siella pneumonia and Pseudomonas aeruginosa) bacteria. Toxicactivity (LC50 of 154.16 g/mL) of MvRL was detected throughbioassay with Artemia salina. In conclusion MvRL is a lectin withantifungal activity and the effect on F. oxysporum f.sp. lycopersiciwas different for all three races. Toxicity and effect of MvRL onFusarium species are indicative of its potential biotechnological useas antifungal and antitumor biomaterial.

doi:10.1016/j.jbiotec.2010.09.662

[P-M.75]

Antibacterial activity of easily purified lichen lectin from Clado-nia verticillaris

D.B.M. Ramos 1, F.S. Gomes 1, P.M.G. Paiva 1, M.D.C. Silva 2, L.C.B.B.Coelho 1,∗

1 Universidade Federal de Pernambuco, Brazil2 Universidade Federal Rural do Semi-Árido, BrazilKeywords: Lichen lectin; Cladonia verticillaris; Antibacterial activity

Antimicrobial proteins, among them the lectins, can be used astools against microbial infections. Lectins constitute a heteroge-neous protein group of non-immune origin, containing two or morebinding sites to carbohydrates or glycoconjugates. Interactionsbetween lectins and bacteria occur through selective agglutination