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cell surface protein and carbohydrate components. Significant co-flocculation occurred when 24h-cells deflocculated by chemicalmodification of cell surface protein component and the 24h-cellsdeflocculated by sodium periodate-oxidation were mixed. Thesefindings suggest that both cell surface protein and carbohydratecomponents play important parts in the intercellular interac-tion of the flocculent 24h-cells. Strong co-flocculation occurredwhen non-flocculent 120h-cells and flocculent 24h-cells weremixed. Treatment with proteolytic enzymes and chemical mod-ification of cell surface protein of non-flocculent 120h-cells didnot affect co-flocculation with flocculent 24h-cells but sodiumperiodate-oxidation deprived 120h-cells of co-flocculent abilitywith 24h-cells. The 24h-cells deflocculated by treatment with pro-teolytic enzymes or chemical modification of cell surface proteinfailed to co-flocculate with untreated non-flocculent 120h-cellswhile the 24h-cells deflocculated by sodium periodate-oxidationco-flocculated with untreated non-flocculent 120h-cells. Addi-tion of cycloheximide into growing culture 24 h after inoculationdepressed the decrease of floc-forming ability. These finding sug-gest that 24h-cells flocculate by interaction between cell surfaceprotein and carbohydrate (mannan) and 120h-cells fail to flocculatebecause of removal of protein component essential for flocculation.

1) J. Inst. Brew., 108, 187-192 (2002).

doi:10.1016/j.jbiotec.2010.09.810

[P-S.4]

Signalling Pathways and Cellular Effects Activated in Endothe-lial Cells by Exogenous AIMP1

V.C. Jackson ∗, S. Dewilde, A. Giuliano Albo, D. Corpillo, K. Lis, B.Canepa

LIMA, BioIndustry Park del Canavese S.p.A., Colleretto Giacosa (TO),ItalyKeywords: AIMP1, Angiogenesis, Endothelial cells, Signalling path-way, Phosphorylation, phosphoproteomics, VEGF, Receptor

AIMP1 (ARS-interacting multifunctional protein 1) is a cofactorof the Aminoacyl-tRNA synthetase complex, but also the precur-sor of EMAP II (Endothelial Monocyte-Activating Polypeptide II), socalled for its ability to act on endothelial cells and on many immunesystem cells (Quevillon et al., 1997). In particular stress conditions,both AIMP1 and EMAP II are found in the extracellular environ-ment, where they show cytokine properties, acting on many celltypes including endothelial cells, immune cells and fibroblasts (VanHorssen et al., 2006; Kao et al., 1992; Berger et al., 2000).

We studied the mechanisms of action of AIMP1 when releasedin the extracellular environment, focusing our attention on signalpathways activated in Porcine Aortic Endothelial Cells by exoge-nous AIMP1.

To do this, different techniques were employed includingbiological assays, immunofluorescence technique to study theentry of exogenous AIMP1 and its subcellular localization, a 2DE-phosphoproteomic analysis to reveal the signalling pathwaysresponsible for the cellular effects and qPCR to analyse transcrip-tional regulated genes.

AIMP1 has a dose-dependent role in angiogenesis, inhibitingECs proliferation at high concentrations and stimulating it at lowconcentrations (Park et al., 2002).

Immunofluorescence studies showed that exogenous AIMP1 isinternalized in ECs and accumulates in clots at the cell membranelevel.

AIMP1 activates ERK and JNK, but not AKT, through phosphory-lation after 10 minutes of treatment compared with a control. Using

a 2DE-phosphoproteomic approach, we confirmed the phosphory-lation of ERK upon AIMP1 treatment and identified several otherputative signalling molecules involved.

Preliminary results of qPCRs performed on a panel of genesfollowing ECs treatment with exogenous AIMP1 showed downreg-ulation of VEGF-A and VEGFR2.

In conclusion, treatment of ECs with exogenous AIMP1 leads toits cell entry, rapid phosphorylation of ERK and JNK and transcrip-tional regulation of the members of the VEGF pathway.

We hypothize that AIMP1 acts on PAE cells through a mem-brane receptor and we are currently trying to identify interactingmembrane proteins through affinity purification.

References

Quevillon, S., Agou, F., Robinson, J.C., Mirande, M., 1997. The p43 component ofthe mammalian multi-synthetase complex is likely to be the precursor ofthe endothelial monocyte-activating polypeptide II cytokine. J Biol Chem 272,32573–32579.

Van Horssen, R., Eggermont, A.M.M., ten Hagen, T.L.M., 2006. Endothelial monocyte-activating polypeptide-II and its functions in (patho)physiological processes.Cyt. Gr. Fact. Rev. 17, 330–348.

Kao, J., Ryan, J., Brett, G., Chen, J., Shen, H., Fan, Y.G., et al., 1992. Endothelialmonocyte-activating polypeptide II. A novel tumor-derived polypeptide thatactivates host-response mechanisms. J. Biol. Chem. 267, 20239–20247.

Berger, A.C., Tang, G., Alexander, H.R., Libutti, S.K., 2000. Endothelial monocyte-activating polypeptide II, a tumor-derived cytokine that plays an important rolein inflammation, apoptosis, and angiogenesis. J Immunother 23, 519–527.

Park, S.G., Kang, Y.S., Ahn, Y.H., Lee, S.H., Kim, K.R., Kim, K.W., et al., 2002. Dose-dependent biphasic activity of tRNA synthetase-associating factor, p43, inangiogenesis. J. Biol. Chem. 277, 45243–45248.

doi:10.1016/j.jbiotec.2010.09.811

[P-S.5]

Benchmarking Spain’s Biotechnology: a comparative perspec-tive

Isidre March-Chorda 1,∗, Jorge Niosi 2, Rosa M. Yagüe-Perales 1

1 University of Valencia, Spain2 Université de Québec à Montréal, CanadaKeywords: Spain; Venture; Patenting; Spin-off

Purpose of this study is to ascertain how different or similar isthe national system of Spanish biotechnology compared to those ofthe leading countries such as Canada, the United Kingdom or theUnited States.

The study is based on both public figures and a databasepurposely built on Spanish dedicated biotechnology firms. The sta-tistical method applied is the ANOVA analysis.

Main findings and results include:Older and larger companies linked with larger domestic groups

(mainly food, pharmaceutical or chemical) request patents mostoften.

Venture capital in Spain has eluded biotechnology and the lastfigures for 2008 and 2009 reveal a cut in the funding of biotechstart-ups.

In Spain, university spin-offs are younger than the average com-pany, and less likely to have venture capital and patents. They arenot particularly aiming at human health products, and are moreoften located in non core regions.

A massive 80% of the growing companies are older than theaverage, and 65% of them are research spin-offs. Growing firmstend to concentrate in a few larger metropolitan areas, while theirPatenting activity is not more intense than the average company.

Growing firms are more often owned by, or affiliated with, agroup of companies. Companies owned by individuals are almostalways struggling. For a vast majority of Spanish firms, human

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S513

health is the application area of nearly three quarters of growingfirms.

To conclude, Spain is entering the biotechnology trail, with agrowing scientific base, a reluctant venture capital industry, andundersized intellectual property management capacity. The firstchallenge is to stimulate private investment in biotechnology eitherby venture capital or by institutional investors. Another challengeis to convert intellectual results stemming from academic researchinto products and processes.

doi:10.1016/j.jbiotec.2010.09.812

[P-S.6]

Rules for computational design of enzymes to increase both thethermostability and the activity

Y.J. Yoo 1,∗, J.C. Joo 1, S.Y. Hong 1, H.J. Park 1, H.S. Yeon 1, S.P. Pack 2

1 Seoul National University, Republic of Korea2 Korea University, Republic of KoreaKeywords: Cavity design; Enzyme flexibility; Spring model;Enzyme engineering

Enzyme stability and activity are important characteristics inthe industrial applications. Recently, much attention has beenpaid on computational design to improve the thermostabilityof enzymes with the accumulated information of relationshipbetween structure/sequence and the thermostability. Despitethe successful enhancement of the thermostability, engineeredmutants often showed activity and the engineering on the enzymeactivity still remains unsolved. It is thus important to developthe general strategies to increase thermostability and activitysimultaneously for engineering enzymes suitable for industrialapplications. Here, we developed computational design strate-gies to improve the thermostability without the reduction of theenzyme activity.

Protein cavities were engineered with analysis of their flexibil-ity to improve the thermostability of enzymes. The results from ourcomputational design increased thermostability and activity at thesame time. Moreover, the spring model based on differences of theflexibility and structures between apo and holo enzymes was pro-posed to predict the residues involved in the enzyme activity. Theresidual forces between apo and holo structures of T4 lysozyme andCandida antarctica lipase B with published experimental data wereanalyzed to validate our spring model. The catalytically importantresidues were predicted by the spring model and successfully engi-neered to improve the enzyme activity and thermostability. And itwas attempted to increase enzyme activity in organic solvents tomodulate enzyme flexibility using spring model. The details will bepresented and discussed.

doi:10.1016/j.jbiotec.2010.09.813

[P-S.7]

Toward Biosynthesis of a Potent Olive Antioxidant: Integrat-ing Protein Engineering and Statistical Methods for ImprovedEnzyme Performance

Moran Brouk 1,∗, Yuval Nov 2, Ayelet Fishman 1

1 Department of Biotechnology and Food Engineering, Technion-IsraelInstitute of Technology, Haifa, Israel2 Department of Statistics, University of Haifa, Haifa, IsraelKeywords: Biocatalysis; Protein engineering; Hydroxytyrosol;Toluene monooxygenases

In recent years, biocatalysis has become an increasinglyimportant technology implemented in pharmaceutical, food, envi-ronmental and chemical industries. The goal of this research isto engineer toluene-monooxygenases, for the environmentally-friendly biosynthesis of the commercially-valuable hydroxytyrosol(HTyr) from the abundant and inexpensive 2-phenylethanol (PEA).

HTyr, one of the most important phenols present in olives,stands out as a compound of high added value due to its excep-tional antioxidant, antimicrobial and anti-carcinogenic activities.The vast amount of data accumulated regarding the benefits of HTyrmakes it a good candidate for pharmaceutical or food preparations(i.e. functional foods). Despite the great potential of HTyr, its com-mercial availability is limited. Therefore, its production will haveimportant industrial applications.

Escherichia coli cells manipulated to express toluene-monooxygenases are capable of oxidizing a wide range ofsubstituted aromatic and phenolic compounds with highregiospecificity. Despite the resemblance of PEA to the natu-ral substrate, toluene, it was found to be a very poor substratefor the wild-type enzymes. In this research, by employing severalprotein engineering approaches, the substrate specificity andoxidation activity of toluene-4-monooxygenase from Pseudomonasmendocina KR1 were dramatically improved. Moreover, a statisti-cal model was developed to give predictions to which mutationsshould be combined for further rise in activity. One triple mutantsuggested by this model, had a 200-fold improvement in activitycompared to the wild-type enzyme. It was concluded that increas-ing the size of the active site pocket and the tunnel entrance,enables for the first time, HTyr formation, which the wild-typeenzyme was not capable of producing. Accordingly, a combinationof random and rational mutagenesis, along with statistical analysisto reduce labor-intensive screening, is an excellent approach forproducing highly active biocatalysts.

doi:10.1016/j.jbiotec.2010.09.814

[P-S.8]

Production of plant cell wall degrading enzymes by the exo-1mutant and wild type Neurospora crassa

A.M. Polizeli 1,∗, M.A. Moraes 2, J.A. Jorge 3, H.F. Terenzi 3, M.L.T.M.Polizeli 3

1 São Paulo University, Departamento Química, FFCLRP/USP, Brazil2 São Paulo State University;Julio de Mesquita Filho, DepartamentoFitotecnia, UNESP, Ilha Solteira, Brazil3 São Paulo University, Departamento Biologia, FFCLRP/USP, BrazilKeywords: exo-1 mutant; Neurospora crassa; avicelase; car-boxymethylcellulase; xylanase; pectinase

The biological decomposition of plant cell wall components is animportant process for biomass degradation. In nature, components