Organic solvents

Biocatalysis in organic solventsBiocatalysis in organic solvents

Improving enzymes by using them in organic solvents

• Alexander Klibanov

• NATURE (2001) 409: 241-246

www.nature.com

Biocatalysis in organic solvents 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


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


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


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


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


Indicators of solvent hydrophobicity

Table 5.2

• Dielectric constant• Dipole moment• Polarizability

• Molar heat of vaporization (Hildebrand solubility)

• Dye solvatochromism

• Log P



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



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



• 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



• 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



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)


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


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


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


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


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



• 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



• 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



• 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



• 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


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


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


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


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


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


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


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


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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Organic solvents

Engineering