Biocatalysis in organic solvents

1. Biocatalysis in organic solvents Improving enzymes by using them in organic solvents • Alexander Klibanov • NATURE (2001) 409: 241-246 www.nature.com

2. Biocatalysis in organic solvents • 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents Indicators of solvent hydrophobicity Table 5.2 • Dielectric constant • Dipole moment • Polarizability • Molar heat of vaporization (Hildebrand solubility) • Dye solvatochromism • Log P

8. Biocatalysis in organic solvents

9. Biocatalysis in organic solvents 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 in organic solvents

11. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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 in organic solvents

15. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents Two-phase systems • [S] low, limits rate of catalysis • Product more hydrophobic than substrate: shift in equilibrium towards product side • Interfacial areais small: limits mass transport • Agitation causes dispersion of organic solvent in aqueous phase: enzyme inactivation

19. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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. Biocatalysis in organic solvents 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