Biocatalysis in organic solvents

1. Biocatalysis in organic solvents Improving enzymes by using them in monophasic organic solvents • Alexander Klibanov MIT, Cambridge, USA • NATURE (2001) 409: 241-246

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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?

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. Biocatalysis in organic solvents Influence diëlectric constant • Low   reduced enzyme flexibility • Medium  optimal flexibility and activity • High  polar solvent strips enzyme water layer

17. 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

18. 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

19. 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 !

20. 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

21. 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

22. 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

23. 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

24. 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