1. Přednáška 1 Imobilizované biologické systémy

2. Immobilized Biocatalysts Doc. Ing. Jiří SAJDOK,CSc.

3. 1.Introduction Working Party on Applied Biocatalysts within the European Federation of Biotechnology: “Immobilized biocatalysts are enzymes, cells, or organelles (or combinations of them) which are in a state that permits their reuse” Working Party on Applied Biocatalysts within the European Federation of Biotechnology: “Immobilized biocatalysts are enzymes, cells, or organelles (or combinations of them) which are in a state that permits their reuse” P. B. Poulsen, Enzyme Microb. Technol. 5 (1983) 304 – 307 P. B. Poulsen, Enzyme Microb. Technol. 5 (1983) 304 – 307

4. Historical Background: ( 1823 vinegar production, sludge, attachment to equipment ) 50s – 60s : immobilization of enzymes ( 1916 Nelson – Griffin: invertase ads.on charcoal 1948 Sunmer: jack bean urease) 1950 – 1970: intensive investigations on immobilized enzymes and other proteins ( e.g.antigens -> affinity chromatography ) ( e.g.antigens -> affinity chromatography ) 1969 – first industrial appt.of immobilized enzyme Optical resolution of DL aminoacids with immobilized amino acylase ( Chibata et al. ) Since 1960 investigations on immobilized cells Industrial applications of immobilized microbial cells: 1973L – aspartic acid -Escherichia coli (aspartase ) 1974 L - malic acid – Brevibacterium ammoniagenes ( fumarase) 1982L – alanin – Pseudomonas dacunhae ( L-aspartate  - decarboxylase )

5. Introduction Biocatalysts dissolved in aqueous buffer solutions Biocatalysts dissolved in aqueous buffer solutions soluble or native enzymes, cells, cell parts, or organelles. soluble or native enzymes, cells, cell parts, or organelles. Immobilized, fixed, or insolubilized enzymes, cells, etc., denote biocatalysts that are bound to a support. Immobilized, fixed, or insolubilized enzymes, cells, etc., denote biocatalysts that are bound to a support. carrier, support, or matrix carrier, support, or matrix cross-linking agent, bifunctional agent, or carrier activator. cross-linking agent, bifunctional agent, or carrier activator.

6. Membránové bílkoviny

7. 2.2. Methods of Enzyme Immobilization 1. modified into a water-insoluble form, 2. retained by an ultrafiltration membrane inside a reactor, or 3. bound to another macromolecule to restrict their mobility.

8. Imobilization Techniques Figure 1. Classification of enzyme immobilization methods Figure 1. Classification of enzyme immobilization methods

10. Encapsulation of Enzyme

11. 2.2.1. Carriers for Enzyme Immobilization 1. Large surface area and high permeability 2. Sufficient functional groups for enzyme attachment under nondenaturing conditions 3. Hydrophilic character 4. Water insolubility 5. Chemical and thermal stability 6. Mechanical strength 7. High rigidity and suitable particle form 8. Resistance to microbial attack 9. Regenerability 10. Toxicological safety 11. Low or justifiable price 1. Large surface area and high permeability 2. Sufficient functional groups for enzyme attachment under nondenaturing conditions 3. Hydrophilic character 4. Water insolubility 5. Chemical and thermal stability 6. Mechanical strength 7. High rigidity and suitable particle form 8. Resistance to microbial attack 9. Regenerability 10. Toxicological safety 11. Low or justifiable price

12. Table 2. Chemical classification of matrixes used for enzyme immobilization Synthetic polymers Synthetic materials Polystyrene Nonporous glass Polyacrylates and poly- Controlled pore glass methacrylates Controlled pore metal Polyacrylamide oxides Hydroxyalkyl methacrylates Metals Glycidyl methacrylates Maleic anhydride polymers Vinyl and allyl polymers Polyamides Natural polymersMinerals Polysaccharides Attapulgite clays Cellulose Bentonite Starch Kieselghur Dextran Pumice stone Agar and agarose Alginate Carrageenan Chitin and chitosan Proteins Collagen Gelatin Albumin Silk Carbon materials (activated carbon)

13. Agar Agar, also called agar-agar, kanten, or gelose, is the oldest known gel-forming polysaccharide Discovered in the 17th century in Japan and consumed for 200 years, agar is extracted from certain marine red algae of the class Rhodophyceae mainly from Gelidium and Gracilaria species, growing essentially along the coasts of Morocco, Spain, Portugal, Chile, Japan and Korea

14. Origin of seaweed extracts — general classification Origin of seaweed extracts — general classification a Species of economic significance b Contains only component mentioned c Contains predominantly underlined component

15. Agar Koch and Petri in 1882 - medium in which to grow bacteria no better solidifying agent in microbiological media has been found microbiological, biotechnological, and public health laboratories, and an important colloid in other industries permitted gelling, stabilizing, and thickening agent for food applications, authorized in all countries without limitations of daily intake (confectionery, bakery, pastry, beverage, sauces, wines, spreads, spices and condiments, meats and fishes, dairy, jams, etc.) Apart from its ability to gelify aqueous solutions and produce gel without the support of other agents, agar can also be used as a safe source of dietary fiber since it is not digestible by the human body.

16. Agar Flow sheet of traditional agar extraction ExtractionPurificationDehydratation

17. Structure of agar agarose 1,4-linked 3,6-anhydro- a- l-galactose alternating with 1,3-linked b-d- galactose Agaropectin repeating unit as agarose, some of the 3,6-anhydro-l- galactose residues can be replaced with l-galactose sulfate residues and the d- galactose residues are partially replaced with the pyruvic acid acetal 4,6-O- (1-carboxyethylidene)-d- galactose

18. Agarosa Processing to remove SO 3 NaBH 4/ -OH Macropourus, hydrophylic Comercialy availability Chemically stable Low non-specific binding Resistent to MO

19. Agarosa

20. Agarosa Zlepšení mechanických vlastností Prokřížení např. epichlorhydrinem

21. Gelling mechanism Hysteresis of 1.5 % agar gels Hysteresis of 1.5 % agar gels Three equatorial hydrogen atoms of the 3,6-anhydro- a-l- galactose residue are responsible for constraining the molecule so as to form a helix with a threefold screw axis Gel formation mechanism in aqueous agar solutions

22. Quick Soluble Agar Comparison of production processes of traditional agar and QSA Patent manufacturing process without any chemical or genetic modifications