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Immobilized Biocatalysts: Methods, Carriers & Applications for Enzyme Immobilization

This lecture provides an introduction to immobilized biocatalysts, their historical background, methods of enzyme immobilization, and various carriers used for immobilization. It also discusses the use of agar as a stable and versatile matrix for enzyme immobilization.

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Immobilized Biocatalysts: Methods, Carriers & Applications for Enzyme Immobilization

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  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” • 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 ) 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: 1973 L – aspartic acid -Escherichia coli (aspartase ) 1974 L - malic acid – Brevibacterium ammoniagenes ( fumarase) 1982 L – alanin – Pseudomonas dacunhae ( L-aspartate  -decarboxylase )

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

  6. Membránové bílkoviny

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

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

  9. Encapsulation of Enzyme

  10. 2.2.1. Carriers for Enzyme Immobilization 1. Large surface area and high permeability2. Sufficient functional groups for enzyme attachment under nondenaturing conditions3. Hydrophilic character4. Water insolubility5. Chemical and thermal stability6. Mechanical strength7. High rigidity and suitable particle form8. Resistance to microbial attack9. Regenerability10. Toxicological safety11. Low or justifiable price

  11. Table 2. Chemical classification of matrixes used for enzyme immobilization

  12. Agar Agar, also called agar-agar, kanten, or gelose, is the oldest known gel-forming polysaccharideDiscovered 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

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

  14. 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 animportant 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.

  15. Agar Flow sheet of traditional agar extraction Extraction Purification Dehydratation

  16. 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

  17. Agarosa Processing to remove SO3 NaBH4/-OH Macropourus, hydrophylic Comercialy availability Chemically stable Low non-specific binding Resistent to MO

  18. Agarosa

  19. Agarosa Zlepšení mechanických vlastností Prokřížení např. epichlorhydrinem

  20. Gelling mechanism 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

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

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