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Immobilized biocatalysts

Immobilized biocatalysts. Enzymes or whole cells physically confined or localised in a certain defined region of space with retention of their catalytic activities and which can be used repeatedly and continuously. Immobilized biocatalysts. Composite of two essential components

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Immobilized biocatalysts

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  1. Immobilized biocatalysts Enzymes or whole cells • physically confined or localised in a certain defined region of space • with retention of their catalytic activities • and which can be used repeatedly and continuously

  2. Immobilized biocatalysts Composite of two essential components • Carrier, designed to aid separation and reuse of the catalyst and facilitates control of the process • Enzyme, designed to convert the substrates of interest into the desired products

  3. Methods of immobilization Figure 3.1 • Binding to a solid support • Cross-linking • Entrapment • Covalent, ionic, hydrophobic binding (Fig. 3.2) • Influence of enzyme structure and behaviour

  4. Colloidal enzymaticnanoreactors Macromol Biosci (2004) 4:13-16

  5. Spherical polyelectrolyticbrush Macromol Biosci (2004) 4:13-16

  6. Preparation of apoflavoprotein Eur J Biochem (2003) 270: 4227-4242

  7. Support materials • large surface area • functional groups • insolubility in water • chemical stability • stability against microbial attack especially important for industrial biocatalysts

  8. Size and shape of insoluble carriers bead fibre capsule film membrane

  9. Selection criteria for carriers • Reactor configurations (batch, stirred-tank, column, plug-flow) • Type of reaction medium (aqueous, organic solvent, two-phase system) • Reaction system (slurry, liquid-to-liquid, liquid-to-solid, solid-to-solid) • Process conditions (pH, temp, pressure)

  10. Aim of chosen parameters • Easy separation of the immobilised enzymes from the reaction mixture • Flexibility of reactor design • Broad applicability in various reaction media and reaction systems • Facilitate down-stream processing and, in particular, control of the process.

  11. Other requirements for ideal BIOCAT • Recyclable • Cost effective • Safe for use • Competitive and innovative enough to protect the intellectual property right • Attractive for end-users

  12. Criteria for robust IMBIOCATs

  13. Optimization of an industrial BIOCAT Glutaryl acylase • stabilisation of the enzyme by multipoint covalent attachment onto a new amino-epoxy Sepabead • parameters that effect the enzyme-support interaction • J Biotechnol (2004) 111: 219-227

  14. Immobilised environment Environment different from solution • effects on [S], pH or ionic strength • restricted diffusion • influence on enzyme kinetic properties • influence on enzyme regio- and enantioselectivity • immobilised enzyme molecules not all identical

  15. Immobilised enzymes Conformational and steric effects • Location of active site • Conformational changes • Partial inactivation by covalent attachment • Reduced number of active molecules • Reduced flexibility

  16. Action of immobilized enzymes Effects on enzyme kinetics • Conformational and steric effects • Partitioning effects (charge) • Micro-environmental effects on intrinsic catalytic parameters (ionic strength) • Diffusional limitation and mass-transfer

  17. -Lactamase Biotin-derivatized PEG-coated sensor chip • Study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing • Sequential adsorption of avidin and biotinylated -Lactamase or immobilisation of preformed complex • Langmuir (2004) 20: 10464 -10473

  18. Enzyme kinetics Chymotrypsin • Acyl-enzyme intermediate • E + S  ES  EA + P1  H2O  E + P2 • Hydrolysis (deacylation) rate limiting step

  19. Chymotrypsin Ester hydrolysis • k2>> k3kcat~ k3 • Km = k3k-1 / k2k1 Amide hydrolysis • k3>> k2kcat~ k2 • Km = k-1 / k1

  20. Chymotrypsin Partitioning effects • Charged matrix can change local pH • pH activity optimum of chymotrypsin (Fig. 3.8) • Anionic polymer: higher pH optimum • Cationic polymer: lower pH optimum • Effects dependent on ionic strength (Fig. 3.9)

  21. Enzyme kinetics Partitioning effects • Attraction or repulsion of charged substrates • Change in local concentration S (PS) • Same charge: Km(app) higher • Opposite charge: Km(app) lower (Fig. 3.10)

  22. Chymotrypsin Micro-environmental effects • Polyanionic EMA-chymotrypsin: higher kcat • Polycationic PO-chymotrypsin: lower kcat (Fig. 3.11) • Perturbation of kcat greater with amides than with esters: acylation step more strongly affected (Table 3.1)

  23. Immobilized chymotrypsin Micro-environmental effects Chymotrypsin copolymers • Amide substrates: similar Km(app) (Table 3.1) • Ester substrates: perturbed Km(app) • Diffusion limitations (effective [S] ) • Change in ratio k3 / k2 and not in k-1/ k1

  24. Immobilised enzymes Micro environmental effects Increase ionic strength • Increase kcat native enzyme • Increase kcat PO-chymotrypsin • No change kcat EMA-chymotrypsin • Change in charge-charge interaction in active site before the acylation step

  25. Immobilised enzymes Mass transfer effects • Rate of substrate diffusion lower than rate of catalysis •  , effectiveness factor: vimm / vsolution dependent on [S], LB plot not linear • External and internal diffusion limitation

  26. Immobilised enzymes Mass transfer effects • External diffusion limitation: Reduced substrate transport from bulk solution to biocatalyst surface • Internal diffusion limitation: Slow diffusion inside porous medium where the enzyme is immobilised in (substrate size)

  27. Applications Dye decolorization • immobilised laccase enzyme reactor • on-line spectroscopy Biotechnol Bioeng (2004) 87: 552-563

  28. Applications Full hydrolysis of lactose in milk • immobilization of lactase from K. lactis • greatly reduces the inhibition by glucose Biotechnol Prog (2004) 20: 1259-1262

  29. Applications Xanthine oxidase • binding to heparin-Sepharose 6B • limits inhibition by clinical relevant inhibitor oxypurinol J Biol Chem (2004) 279: 37231-37234

  30. IMBIOCAT and process development

  31. Carrier-bound enzymes Savings • Enzyme re-use • Downstream processing Additional costs • Reaction times (lower activity and productivity) • Immobilisation process (laborious design)

  32. Carrier-bound or carrier-free Cross-linking • CLE cross-linked dissolved enzyme • CLEA cross-linked enzyme aggregate • CLEC cross-linked enzyme crystal • CLSD cross-linked spray-dried enzyme Curr Opin Biotechnol (2003) 14: 387-394

  33. Carrier-free immobilised enzymes

  34. CLECs Highly active and stable immobilised enzymes of controllable size • Selection of right crystal form or size • Engineering properties crystallization medium • Activity dependent on size and properties substrates, reaction medium, reaction type and reaction conditions Chemtech (1997) 27: 38-45

  35. CLEAs Preparation, optimization and structures • simplicity of operation • no need for laboriuos optimization • no need for pure enzymes • high-throughput methodologies • high yield of activity for any enzyme Biotechnol Bioeng (2004) 86: 273-276

  36. Activity retention and enzyme solubility

  37. CLEAs: current research topics Particle size and diffusion constraints • Broad range of enzymes • Size control • New aggregation methods • New cross-linkers Biotechnol Bioeng (2004) 86: 273-276

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