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IMMOBILIZATION التقييد Dr. Cheba Ben Amar Dept .of Biotechnology, Faculty of Nature and Life Sciences, (USTOMB), Oran, Algeria  


INTRODUCTION The accumulating knowledge in nowadays biotechnology led to widespread uses of biocatalysts and enzymes especially in many fields ranging from waste treatments and agrochemicals to food and biomedical industries. However, most enzymes are relatively unstable ; their purification and recovery in active form still difficult and costs effective [1]

Immobilization definition:

Immobilization definition The term “ immobilized enzymes” refers to “enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously ”

Immobilization history:

Immobilization history Step Date Use Example First 1815 Empirical use in processes acetic acid and waste water treatment . Second 1960s Single enzyme immobilization: production of Laminoacids , isomerization of glucose Third 1985–1995 Multiple-enzyme immobilization including co-factor regeneration and cell immobilization . production of L- aminoacids from keto -acids in membrane reactors

Immobilization history:

Immobilization history The first industrial use of immobilized enzymes was reported in 1967 by Chibata and co-workers, who developed the immobilization of Aspergillus oryzae aminoacylase for the resolution of synthetic racemic D-L amino acids

immobilization advantages:

immobilization advantages -enhancing catalytic activity , pH and thermal stability, reduce biocatalysts washout , increase productivity , facilitate the recovery for continuous operation and repeated use easier product separation, wider choice of reactor improve the mass and heat transfers

immobilization advantages:

immobilization advantages - Furthermore immobilization can also help to enable the employment of enzymes in -different solvents , -at extremes of pH and temperature -and at high substrate concentrations

Immobilized Enzyme Systems Disadvantages:

Immobilized Enzyme Systems Disadvantages Loss or reduction in activity Diffusional limitation Additional cost

immobilization applications :

immobilization applications

immobilization methods:

immobilization methods

Some selection criteria of the immobilization technique.:

Some selection criteria of the immobilization technique . Reaction system Method of immobilisation Dilute aqueous solution Covalent Crosslinking Encapsulation Dilute organic solution Any Concentrated , viscous organic / inorganic mixtures Covalent, crosslinking

immobilization carriers:

immobilization carriers The terms solid phase support, carrier, and matrix are used synonymousl y

Ideal support properties:

Ideal support properties Include physical resistance to compression hydrophilicity inertness toward enzymes ease of derivatization , biocompatibility, resistance to microbial attack , and availability at low cost

Classification of Supports:

Classification of Supports

Natural Organic Supports :

Natural Organic Supports

chitin- chitosan based materials as the most suitable carriers:

chitin- chitosan based materials as the most suitable carriers in general many workers used chitin- chitosan based materials as the most suitable carriers for immobilization regarding to their height affinity to proteins , hydrophilicity, mechanical stability rigidity, regenerability simplicity of preparation the availability of reactive functional groups for direct reactions with enzymes and for chemical modifications .

PowerPoint Presentation:

For all this purposes these natural materials used for immobilization of many enzymes such as lysozyme , cellulase , xylanase, laccase , α-amylase, pectinase , lipase , alkaline protease , keratinase, urease, uricase , tannase and many other enzymes

immobilized enzyme system components:

immobilized enzyme system components

Some effecting parameters in biocatalyst immobilisation:

Some effecting parameters in biocatalyst immobilisation

Selected characteristic parameters of immobilized enzymes:

Selected characteristic parameters of immobilized enzymes Enzyme Biochemical properties molecular mass, prosthetic groups, functional groups on proteinsurface , purity (inactivating/protective function of impurities) Enzyme kinetic parameters ; specific activity, pH-, temperature profiles, kinetic parameters for activity and inhibition, enzyme stability against pH, temperature, solvents , contaminants, impurities Carrier Chemical characteristics chemical basis and composition, functional groups, swelling behavior, accessible volume of matrix and pore size, chemical stability of carrier; Mechanical properties ; mean wet particle diameter, single particle compression behavior,flow resistance (for fixed bed application), sedimentation velocity (for fluidized bed), abrasion (for stirred tanks) Immobilized enzyme Immobilization method ; bound protein, yield of active enzyme, intrinsic kinetic parameters (properties free of mass transfer effects) Mass transfer effects consisting of partitioning (different concentrations of solutes inside and outside the catalyst particles), external and internal (porous) diffusion; this gives the effectiveness in relation to free enzyme determined under appropriate reaction conditions, Stability ; operational stability (expressed as activity decay under working conditions), storage stability ; “Performance” productivity (amount of formed product per unit or mass of enzyme) enzyme consumption (e.g. units kg–1 product, until half-life)

Immobilization products :

Immobilization products The application of immobilized whole cells for the production of metabolites by microorganisms has been extensively studied and several products of value such as alcohols , organic acids , amino acids, antibiotics , steroids, and enzymes

Major Products Obtained Using Immobilized Enzymes:

Major Products Obtained Using Immobilized Enzymes Enzyme Product Glucose isomerase Amino acid acylase Penicillin acylase Nitrile hydratase β- Galactosidase High-fructose corn syrup Amino acid production Semi- synthetic penicillins Acrylamide Hydrolyzed lactose ( whey )

Somme references:

Somme references [1] Brena , B. M. and Batista- Viera , F. Immobilization of Enzymes From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ.2006   [2] Longo, M.A.,Novella , I.S.,.Garcia , L.A and Diaz, M. Diffusion of protease in calicium alginate beads . Enz.Microb.Technol . ; 1992. 14:586-590.   [ 3 ] Hanefeld , U.; Gardossi , L.;and . Magner , E. Understanding enzyme immobilisation ; Chem.Soc.Rev.,2009.38 : 453–468.   [4] Guibault , G. G., Kauffmann, J. M., and Patriarche , G. J. Immobilized Enzyme Electrodes as Biosensors. In: Protein Immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NT, 1991.pp. 209–262.   [5] Taylor, R.F. Immobilized Antibody and Receptor Based Biosensors. In: Protein Immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NY;1991. pp. 263–303.   [6] Chang, M. S. Therapeutic Applications of Immobilized Proteins and Cells.In : Protein Immobilization. Fundamentals and applications (Taylor, R. F., ed.), Marcel Dekker, New York, NY, 1991. pp. 305–318.   [7] Tischer · W., and Wedekind . F. Immobilized Enzymes: Methods and Applications. Topics in Current Chemistry, Vol. 200 © Springer Verlag Berlin Heidelberg 1999 [9] Cheba,B . A. , Zaghloul , T. I. , EL- Mahdy , A. R. ,and EL- Massry , M. H. Enhanced Production of Bacillus sp . R2 Chitinase through Cell Immobilization. ACT-Biotechnology Research Communications, 2011. 1.1:8-13   [8] Cheba , B.A. Chitin and Chitosan: Marine Biopolymers with Unique Properties and Versatile Applications. Global Journal of Biotechnology & Biochemistry.,2011. 6 (3): 149-153. [28] Krajewska , B. Application of chitin and chitosan-based materials for enzyme immobilization: a review. Enz . Microb . Technol. ; 2004. 35: 126-230.

Thank you for your kind interest:

Thank you for your kind interest

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