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biocatalysis in organic solvents
Biocatalysis in organic solvents

Improving enzymes by using them in monophasic organic solvents

  • Alexander Klibanov

MIT, Cambridge, USA

  • NATURE (2001) 409: 241-246
biocatalysis in organic solvents2
Biocatalysis in organic solvents

Principle

  • Enzymes do not notice all water moleculesin solution, just the ones that are nearby
  • Replace bulk water with organic solvent
  • Moist, solid enzyme, suspended in organic solvent
biocatalysis in organic solvents3
Biocatalysis in organic solvents

Advantages

  • Easy downstream processing

Filter off enzyme, evaporate the solvent and separate product from remaining starting material

  • No need for immobilisation
  • Shift thermodynamic equilibria
  • Change enzyme selectivity
biocatalysis in organic solvents4
Biocatalysis in organic solvents

Solid enzymes

  • Catalytically active when water content < 2%

Precautions

  • Stirring, shaking or sonication needed for S-diffusion
  • Optimise micro-pH around enzyme
  • Control water activity
  • Use correct hydrophilicity organic solvent
biocatalysis in organic solvents5
Biocatalysis in organic solvents

Diffusional limitation

  • Crystalline, lyophilized, precipitated or adsorbed enzyme
  • Sufficient mobility required to allow minor conformational changes (formation ES-complex)
  • Substrate channeling occurs when sufficient agitation is provided
biocatalysis in organic solvents6
Biocatalysis in organic solvents

Lyophilization

  • Dehydration may change enzyme structure
  • Use of lyoprotectants, such as sugars, PEG, certain inorganic salts, substrate-resembling ligands and crown ethers
  • Activation up to four orders of magnitude
biocatalysis in organic solvents7
Biocatalysis in organic solvents

Effect of pH Fig. 5.8

  • pH measurements not easy
  • Ionization sate of enzyme determines its conformation, activity and selectivity
  • Employ solid enzymes that have been recovered from lyophilization or precipitation from a buffer at their pH optimum: pH memory
biocatalysis in organic solvents8
Biocatalysis in organic solvents

Effect of water

  • How much water is required to retain catalytic activity?
  • How can we define the amount of water in the reaction mixture?
  • How can we control water activity in the reaction mixture?
biocatalysis in organic solvents9
Biocatalysis in organic solvents

How much water is required to retain catalytic activity?

  • Enzyme dependent (chymotrypsin, tyrosinase)
  • Tightly bound water remains present, even after lyophilization
  • Often an optimal water content can be found

Fig. 5.9

biocatalysis in organic solvents10
Biocatalysis in organic solvents

Reaction rate vs. stability

  • Low rate at low water content Fig. 5.9
  • Stability high at low water content Fig. 5.10
  • Water is involved in inactivation reactions
  • Water increases enzyme flexibility  unfolding
biocatalysis in organic solvents11
Biocatalysis in organic solvents

How can we define the amount of water in the reaction mixture?

  • Concentration or volume percentage not useful
  • Degree of hydration (enzyme bound water)
  • Thermodynamic water activity aw
biocatalysis in organic solvents12
Biocatalysis in organic solvents

Water activity

  • Pv (water-solvent) / Pv (water-water)
  • Determines how much water is bound to the enzyme
  • Determines the catalytic activity to a great extent
  • Determines the effect of water on the chemical equilibrium position
biocatalysis in organic solvents13
Biocatalysis in organic solvents

Fixed water activity

  • Allows to investigate the influence of solvent on enzyme catalysis
  • Corrects for different degree of hydration due to different solubility (cf. hexane - ethyl acetate)
  • At a known water activity the hydration of enzyme is fixed
biocatalysis in organic solvents14
Biocatalysis in organic solvents

How can we control water activity in the reaction mixture?

  • Pre-equilibration of both enzyme and substrate solution in atmospheres of controlled water activity (before mixing)
  • Range of water activities can be obtained by using different saturated salt solutions
biocatalysis in organic solvents15
Biocatalysis in organic solvents

The nature of the organic solvent

  • Apolar water-immiscible solvents (log P > 4)
  • Laane et al. 1987 Fig. 5.11
  • No clear correlation in the region 1 < log P < 4
  • Influence diëlectric constant Fig. 5.12
biocatalysis in organic solvents16
Biocatalysis in organic solvents

Influence diëlectric constant

  • Low   reduced enzyme flexibility
  • Medium  optimal flexibility and activity
  • High  polar solvent strips enzyme water layer
biocatalysis in organic solvents17
Biocatalysis in organic solvents

Hydrophobic active sites like hydrophobic substrates

  • Rate enhancement of enzymes in waterbecause the substrate wants to partition from water into the active site
  • In organic solvent the substrate is no longer squeezed out of the medium owing to the hydrophobic effect and the energetic advantages of partioning drop lower rates
biocatalysis in organic solvents18
Biocatalysis in organic solvents

Energy of the transition state

  • Many enzymes form charged tetrahedral reaction intermediates (polar transition state)
  • Stabilised by internally bound water
  • Shielded from solvent
  • Diminished activity in organic solvent
biocatalysis in organic solvents19
Biocatalysis in organic solvents

Probing of biochemically significant enzyme intermediates

  • Generation and stabilization of horseradish peroxidase compound II in neat benzene solution

at room temperature

  • Patricia Mabrouk JACS (1995) 117, 2141-2146
  • PEGylated enzyme: highly soluble !
biocatalysis in organic solvents20
Biocatalysis in organic solvents

Effects on enzyme selectivity

  • Change in substrate, enantiomeric, prochiral, regio- and chemoselectivities
  • Substrate recognition
  • Proteases: driving force of enzyme-substrate binding is hydrophobic interactions between the side chain of amino-acid substrate and enzyme active site
biocatalysis in organic solvents21
Biocatalysis in organic solvents

Effects on regio-and chemoselectivity

  • Lipases
  • Preference for distinct functional group in substrate molecule
  • Acylation of hydroxyl group in relation to amino group favored in organic solvent
biocatalysis in organic solvents22
Biocatalysis in organic solvents

Effects on enzyme selectivity

  • Change in product specificity
  • Hydroxylation efficiency of vanillyl-alcohol oxidase with 4-alkylphenols
  • Organic solvent influences access of water to the active site
biocatalysis in organic solvents23
Biocatalysis in organic solvents

Medium engineering

Tailoring the medium composition to optimise

the reaction yield

  • Effects on enzyme activity and stability
  • Requirements for water, pH and buffer capacity
  • Solubility and stability substrate and products
  • Recovery of products, separation of biocatalyst and removal of substrate
biocatalysis in organic solvents24
Biocatalysis in organic solvents

Protein engineering

Site-directed mutagenesis

Random mutagenesis

  • Minimisation of surface charges, enhancement of internal polar interactions, disulfide bonds etc.
  • Change of substrate specificity, binding affinity or stereospecificity