Organic solvents

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