html5-img
1 / 38

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

salena
Télécharger la présentation

Immobilized biocatalysts

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related