Organic solvents

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  1. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Improving enzymes by using them in organic solvents ã Alexander Klibanov ã NATURE (2001) 409: 241-246…
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  • 1. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Improving enzymes by using them in organic solvents • Alexander Klibanov • NATURE (2001) 409: 241-246 www.nature.com
  • 2. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss • The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than in their natural aqueous reaction media • Enzymes can catalyze reactions impossible in water, become more stable and exhibit behaviour such as molecular memory • Enzymatic selectivity can be markedly affected
  • 3. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Water is a poor solvent in preparative organic chemistry • Insolubility, decomposition of reagents • Large scale removal of water is tedious and expensive due to its high boiling point and high heat of evaporation • Side reactions such as hydrolysis, racemisation and polymerisation
  • 4. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss New enzymatic reactions • Lipases, esterases, proteases ester + water ® acid + alcohol • In anhydrous solvents and by adding alternative nucleophiles such as alcohols, amines and thiols transesterification, aminolysis and thiotransesterification
  • 5. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Systems with organic solvents • Water and a water miscible organic solvent • Two-phase systems • PEG-modified enzymes in organic solvents • Reversed micelles • Monophasic organic solvents
  • 6. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Potential advantages and bottlenecks • Table 5.1 gives a summary of the potential advantages of enzymes in organic solvents • Need for guidelines what system is the best under the given circumstances • Solvent hydrophobicity
  • 7. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Indicators of solvent hydrophobicity Table 5.2 • Dielectric constant • Dipole moment • Polarizability • Molar heat of vaporization (Hildebrand solubility) • Dye solvatochromism • Log P
  • 8. Biocatalysis iinn oorrggaanniicc ssoollvveennttss
  • 9. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Log P Fig. 5.2 P = [X]octanol / [X]water • most widely used indicator of solvent polarity • log P < 2 distortion of water structure • 2 < log P < 4 unpredictable effects • log P > 4 intact water structure
  • 10. Biocatalysis iinn oorrggaanniicc ssoollvveennttss
  • 11. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Effects on enzyme stability • Dry enzymes are not active, but regain their activity when some water is added • Water is needed for flexibility (molecular lubricant) and essential parts of the enzyme surface must be hydrated to allow catalysis • Hydrophobic solvents leave the hydration shell of the protein intact
  • 12. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Effects on enzyme stability • Hydrophobic solvent: small redistribution of water: conservation of native protein structure • Polar solvent: stronger partitioning effect Interaction of solvent with protein surface Strip tightly bound water Destruction of hydrogen bond network Lowering of surface tension Onset of protein unfolding
  • 13. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Effects on enzyme stability • Extreme thermostability in inert solvents Fewer side reactions (deamidation, hydrolysis) Conformational rigidity in dehydrated state • Half-life of enzyme at high temperature drops precipitously when the water content is raised • Chymotrypsin, lipase, ribonuclease
  • 14. Biocatalysis iinn oorrggaanniicc ssoollvveennttss
  • 15. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Water-water miscible solvents • Polar solvents detrimental to enzymes (log P < 1) low concentrations tolerable (10-30%) • Reactant, inhibitor, increase of flexibility (rate) • Operational stability (Table 5.3) • Change in product pattern (Fig. 5.3)
  • 16. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Substrate solubility • Presence of organic solvent can have a large effect on substrate solubility • A substrate with a low affinity for solvent binds strongly to the enzyme • Change in kinetic parameters (Km), S-specificity • Polar substrates have high Km in polar solvent
  • 17. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Two-phase systems • About equal volumes of an aqueous solution and an immiscible organic solvent • Catalysis takes place in the aqueous phase or at the interface • [S] dependent on partition coefficient • Organic phase acts as a substrate reservoir
  • 18. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Two-phase systems • [S] low, limits rate of catalysis • Product more hydrophobic than substrate: shift in equilibrium towards product side • Interfacial area is small: limits mass transport • Agitation causes dispersion of organic solvent in aqueous phase: enzyme inactivation
  • 19. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Two-phase systems • S-specificity and catalytic activity comparable to pure water system • Traces of solvent can influence activity and stability • Enzyme recovery is difficult • Immobilisation allows reuse of biocatalyst
  • 20. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss PEG-modified enzymes • Modification of lysine residues with amphipathic PEG molecules of different size • Fig. 5.5 Synthesis of organic solvent soluble enzymes • Triazine activated PEG2 • Degree of modification can be controlled
  • 21. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss PEG-modified enzymes • 10 - 20 PEG chains per enzyme molecule • Increase in molecular mass • Creation of hydrophilic micro-environment around enzyme molecule • Protects enzyme from surrounding organic solvent and prevents stripping of essential water
  • 22. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss PEG-modified enzymes • Radius hydrophilic environment up to 30 nm due to length of PEG 5000 • High enzymatic activity with water immiscible solvents • Table 5.4 Enzymatic activity in different organic solvents
  • 23. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss PEG-modified enzymes • Improved storage and thermal stability • Modification of kinetic parameters • Modification of S-specificity • Partitioning of apolar substrates is unfavourable • S-diffusion needs to be sufficiently rapid • Hexane or ether precipitation: good recovery
  • 24. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss PEG-modified enzymes • Fe-carboxy-PEG Magnetic beads: easy recovery Cost aspects of biocatalyst preparation • Medical applications Severe combined immunodeficiency (SCID) PEG-ADA stays in the blood for 1-2 weeks Protease-resistant, not excreted by kidney No receptor binding: no immunoresponse
  • 25. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • Form spontaneously when a surfactant is dispersed in an apolar solvent in the presence of a few volume percent of water • Sometimes a cosurfactant (alcohol) is required • Droplet size in the nm range, dependent on w0 • Thermodynamically stable, optically transparant • Ions to proteins can be incorporated
  • 26. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • Collision induces content exchange • Transport between water core and organic phase allows reactions between polar and apolar compounds • Enzyme can be solubilized in different ways: Extraction from dry powder or solvent Injection from concentrated solution
  • 27. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • Enzyme location Fig. 5.6 - in water pool - in contact with surfactant head groups - in between the surfactant layer • Location is dependent on charge surfactant and charge distribution of the protein • Attractive membrane mimetic system
  • 28. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • Effects on enzyme stability - dependent on protein properties - restricted mobility may prevent unfolding - encapsulation limits autolysis of proteases - low water content increases stability
  • 29. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • Effects on enzyme activity - Water content too low - pH different from stock buffer solution due to binding of protons or hydroxyl groups with surfactant head groups - Unsufficient buffer capacity
  • 30. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • Effects on enzyme kinetics - Partitioning effects substrates - Increase in apparent Km - One or a few substrate molecules per micelle - Reversible kinetics (intramicellar [P] high) - Collision induced exchange kinetics
  • 31. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • Several features of interest for applications - Good stability and recovery - Solubilization of apolar compounds - Cofactor regeneration is possible - Major drawback: presence of surfactant - Limits recovery and purification of apolar substances from the organic phase
  • 32. BBiiooccaattaallyyssiiss iinn oorrggaanniicc ssoollvveennttss Reversed micelles • No scale-up information available - Phase diagram sensitive to T and P - Stability in stirred tank or membrane reactor ? - Not suitable for synthetic reactions - Some promise for purification of enzyme from fermentation broth
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