Ebru Toksoy ner IBSB Industrial Biotechnology and Systems Biology - PowerPoint PPT Presentation

Workshop BioGlue Vienna – Nov 2010 Ebru Toksoy Öner IBSB – Industrial Biotechnology and Systems Biology Research Group Marmara University, Department of Bioengineering, Istanbul, Turkey

 IBSB  Extremophiles  Levan by Bacillus sp.  Extremophilic Levan

Industrial Biotechnology and Systems Biology Research Group 1 Post-Doc Ebru Toksoy Oner, Assoc. Prof. 8 pHD students Kazım Yalcın Arga, Assist. Prof. 2 MS students Marmara University Department of Bioengineering http://ibsb. marmara.edu.tr Istanbul, Turkey

Industrial Biotechnology and Systems Biology Research Group Stephen G. Oliver, Prof.  interpret and optimize production capabilities of yeast, bacteria and extremophiles via systems based approach  optimization of fermentation processes to Barbara Nicolaus, Prof. design high-yield production lines  production of biopolymers form extremophiles Robert Dekker, Prof.  isolation and identification of extremophiles  strain improvement for bioethanol production Betül Kırdar, Prof. Marmara University Department of Bioengineering Ion N. Mihailescu, Prof. Istanbul, Turkey http://ibsb. marmara.edu.tr

ecological systems such as hot springs, salt and soda lakes, deserts and ocean beds that are not compatible with human life are considered as being extreme . Extreme conditions can refer to physical extremes (temperature, pressure or radiation) or geochemical extremes (salinity, desiccation, oxygen tension and pH). Extremophiles are microorganisms that no not on only ly tolerate olerate such extreme conditions, but bu usually ly re requ quire ire such environmental extremes for their survival and growth.

Halomonas sp. AAD6 Isolated from Camaltı Saltern Area (Turkey) Anoxybacillus amylolyticus MR3C T Isolated from M. Rittmann (Antarctica) T 60 ° C Geobacillus thermoleovorans subsp. stromboliensis sbsp.nov Isolated from the geothermal volcanic environment (Italy) T 70 ° C

Starchy agro- industrial wastes Bioethanol …

System ems Biolog logy Aprroa oach to success essfully fully enginee eer Biopoly lymer propertie ies and improv ove e productio ion Metabol olic Model of Halomonas s sp. Protein-encoding gene models 584 Metabolites 1389 Intracellular metabolites 1020 Extracellular metabolites 369 Reactions 1080 Enzymatic reactions 870 Transport fluxes 210 Micr crobial l Produ duct ctio ion

Drug Delivery Systems  Levan – based micro/nano particles Chemical media may be a potential delivery system for macromolecules. Nanostructured Coatings for Biomedical Applications  Thin, high-quality and uniform films produced by the MAPLE (Matrix Assisted Pulsed Laser Evaporation) technique Micr crobial l Environmental Applications Produ duct ctio ion  Emulsifying Agent  Bioflocculating activity Functional Biofilms Low cost substrates  Edible Food Packaging

a homopolimer of fructose units (polyfructose, fructan)   a crucial component of drought and freeze protection in plants ability to stabilize membranes in dry and cold environments  FRUCTAN Levan linear β -(2,6)-linked fructofuranosyl units Inulin Graminan linear β -(2,1)-linked fructofuranosyl units complex and branched

Medica cal Foods Fo Indust strial ial Others ers Pharma rmaceut ceutica cal  bioadhesive  Blood plasma extender  Add sweetness to foods  Emulsifier  Provide viscosity  Hypocholesterolaemic agent  prebiotics  Formulation aid  Holding capacity for water  Anti-AIDS agent  Filler  Stabilizer and thickener and chemicals  Immunostimulating agent  Bulking agent  Surface-finishing agent  Selective plugging agent  Tablet binder  Substitute for gums  Encapsulating agent  Source for pure fructose  Animal feed  Cosmetic  cryoprotectant  Edible food packaging  ….  Carrier for flavor and fragrances

 produced by Bacillus sp.  has strong bioadhesive properties  hydroxyl groups in its structure form adhesive bonds with various substrates. BENEF EFITS S :  Produced from renewable resource, sugar. Dr. Joan Combie  No petroleum or natural gas derivatives in product. Montana Polysaccharides Corp. (USA)  No VOCs, HAPs or other toxic emissions.  Water-based with no solvents.  No health issues for users. Natural Polymer Tensile Strength  No dermal irritation. psi  No allergic contact sensitization. Levan 991  Biodegradable. Carboxymethylcellulose 193  Reduce regulatory burden. Inulin 124  Long term storage as powder. Guar gum 63 Xanthan gum 33 http://specialtybiopolymers.com the most promising commercial polysaccharide based adhesives are actually made from levan • Combie, J. (2005) Properties of Levan and Potential Medical Uses, Polysaccharides for Drug Delivery and PharmaceuticalApplications, June 22, 2006. Vol.934, 263-269. • Mancuso Nichols et al. (2009) 'Screening Microalgal Cultures in Search of Microbial Exopolysaccharides with Potential as Adhesives', The Journal of Adhesion, 85: 2, 97 — 125.

 Very limited information and literature  Not produced at large scale  Production conditions depend on the microbial system used  MW and degree of branching depend on the production conditions  Biological activity and physicochemical properties depend on production  Strict control over process parameters is necessary  Difficult to purify  Expensive an optimal cost-effective production process is a must!

• Grasses ( Dactylis glomerata, Poa secunda, Agropyron cristatum ) Plant • Zymomonas mobilis • Bacillus sp. • Erwinia herbicola • Gluconoacetobacter xylinus • Microbacterium laevaniformans • Rahnella aquatilis • Serratia levanicum Microorganisms • Pseudomonas syringae • Halomonas sp. the first and only levan-producer extremophile * Highest production yield on available substrate * Poli A. et al.. 2009. Carbohydrate Polymers, 78, 651-657.

Chemical media + osteoblast cells isolated from mouse monocyte/macrophage cell line J774 the calvaria of Wistar rats sucrose viability/proliferation 1.2 ALP secretion 1 viability/proliferation (OD595nm) ALP secretion (μg/ml) 0.8 0.6 0.4 0.2 0 Control EPS Biocompatibility studies showed that levan produced by Halomonas sp. did not affect cellular viability and proliferation of osteoblasts and 10 X increase in murine macrophages suggesting the high biocompatibility of this EPS. uronic acid content !! Brine Shrimp Test. The inhibition of avarol toxic activity on brine shrimp (Artemia salina) test was performed in artificial sea water. With decreasing doses of levan solution (500, 50, 5 ppm), protective effect against the toxic activity of avarol increased. Halomonas levan was found to increase the LD50 value of avarol from 0.18 ppm up to 10 Microb obial ppm. The protective effect of the polymer against the toxic activity of Production on by Avarol implied its potential use as an anti-cytotoxic agent. Halomonas sp. cancerous cell line He La (human cervical cancer cells) non-cancerous cell line L929 (mouse fibroblast cells) Sugar beet molasses MTT cell proliferation assay was employed to assess the cell viability. Levan by Halomonas sp. showed high biocompatibility and affinity against both cell lines.

Idea dea: extreme conditions (salinity) at which levan is microbially produced may also confer it some unique properties enhancing its adhesive strength ! current research efforts on levan from Halomonas sp. are now focused on elucidating its potential to be used as a commercially useful adhesive by  Developing new formulations with the polymer and its modified forms  Understanding its mechanism of action

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