SEPARATION BY ENZYMATICALLY CATALYZED REACTIONS

1. Chapter 10 SEPARATION BY ENZYMATICALLY CATALYZED REACTIONS

2. Reactive separation Carbon dioxide + r-1-Phenylethylacetate r,s-1-Phenylethanol s-1-Phenylethanol r-1-Phenylethylacetate Catalyst: Novozym 435 (Lipase) Vinylacetate Carbon dioxide, supercritical s-1-Phenylethanol Carbon dioxide

3. Enzyme Catalysis Kinetics: Influence of Substrates and Enzymes Michealis-Menten-mechanism Reaction of 2nd order with an equilibrium in front. Free enzyme and substrate are in equilibrium with the enzyme-substrate-complex. • [E] Enzyme concentration,[S] Substrate concentration,[ES] Concentration of enzyme-substrate-complex,[P] Product concentration.

4. Enzymes in supercritical carbon dioxide • The use of enzymes in non-aqueous media has several reasons: • Hydrolysis is avoided, • Solubility of organic molecules is better, resulting in higher yields, • In supercritical carbon dioxide the removal of reactands is possible during the reaction.

5. Enzymes in supercritical carbon dioxide: Example Randolph investigated the phospholipase (EC 3.1.3.1) catalyzed hydrolysis of Di-Natrium-p-nitrophenylphosphate in supercritical CO2. • Hydrolysis of Di-Natrium-p-nitrophenylphosphate • in supercritical CO2 (p = 10 MPa, T = 308 K).

6. Influence of water on stability and activity For most enzymes: activity increases with increasing water content. part of the water is fixed to the protein by hydrogen bonding as a water shell. enzymes can be active without water in organic media. additional amount of water should be kept low. conformation of the enzyme seems not to depend on acidity.

7. Activity in water and in sc-CO2 Comparison of contact rate of several enzymes in aqueous solution and of alcohol dehydrogenase in scCO2

8. Specific enzymes and substrates Hrnjez et al.: regio specific and stereo specific activity of the lipase-catalyzed esterification of chiral dioles with anhydrous butyric acid. • Lipase-catalyzed esterification of chiral dioles with anhydrous butyric acid. (p = 3.5 - 20 MPa, T = 40 °C).

9. Lipases Lipases split fats into fatty acids and glycerol • Activation of lipases by the surface • between hydrophobic and hydrophilic medium.

10. Enrichment of enantiomers by lipase catalysis Enantio selective synthesis catalyzed by enzymes is due to the faster reaction of one enantiomer. Scheme of a catalyzed enantio-selective transesterification

11. Enantioselectivity kcat/KM apparent equilibrium constant for the 2nd order reaction at infinitesimal substrate concentration, kcat and KM represent the turnover number and the Michaelis-Menten constant. If the Michaelis-Menten-constant is equal for both substrates: Indices A and B stand for the enantiomers.

12. Enantioselectivity For irreversible reactions the enantioselectivity E can be derived from yield (U) and the enantiomeric excess (ee). U Yield [%],eeProdukt Enantiomeric excess of products [%],eeSubstrat Enantiomeric exc. of substrate [%],cProdkukt Product concentration [mol / l],cSubstrat Substrate concentration [mol / l],cR Concentration R-enantiomer [mol / l],cS Concentration S-enantiomer [mol / l],E Enantioselectivity.

13. Enantioselectivity E = Change of enantiomeric excess with product formation.

14. Enantioselectivity E • The highest enantiomeric excess is achieved • (with high enantioselectivity) at 50 %.

15. Enantioselectivity If the reaction proceeds, ester and alcohol react reversibly into the initial substrates (educts). kcat and kr are the rate constants of forward and backward reaction.

16. Enantioselectivity Variation of enantiomeric excess of the remaining substrate with increasing K (K = 0; 0,1; 0,5; 1; 5) for E = 100.

17. Enantioselectivity • Variation of enantiomeric excess with increasing K (K = 0; 0,1; 0,5; 1; 5) for E = 100. • With the formation of products, enantiomeric excess drops rapidly.

18. Separation by enymatically catalyzed reactions: Examples and Experiments

19. Racemic mixtures of Ibuprofen a. epi-Methyljasmonate Enrichment by enzyme catalyzed interesterification in supercritical carbon dioxide

20. Reaction scheme Lipase: Novozym 435 (strain of Candida antarctica), 1-2 % w/w H2O Solvent: Supercritical CO2, Hexane Conditions:100 - 200 bar, 40 - 60 °C

21. Test cell P: up to 4000 bar T: 150 °C. 0.1 mmol ester, 0.4 mmol alcohol 15 mg immobilized lipase High pressure cell

22. Enantiomeric Excess: Ibuprofen p = 100 bar, T = 50 °C; Catalyst: Lipase Novozym 435 product educt Conversion of Racemic Ibuprofenmethylester With Alcohols in n-Hexane

23. Competitive Process: Chromatography Enantiomer separation by chiral Gas chromatography

24. Influence of various reaction partners

25. Enantiomeric excess: Ibuprofen Interesterification of ibuprofen methylester with ethanol (T = 50 °C)

26. Influence of water Reaction rate independent of Enzyme water content

27. Enrichment of (+)-epi-Methyljasmonate in SC-CO2

28. Some conclusions • Below 10 % percent yield the enzyme catalyzed reaction is irreversible. • (R)-Ibuprofenmethylester is preferably converted. •  The enantiomeric excess increases with decreasing pressure and decreasing temperature. • The enzyme specificity is enhanced by introducing polar functional groups into the acyl acceptor. • Optimum water content is 1-2 % w/w. • Addition of water to Novozym 435 does not accelerate the reaction rate. • (+)-epi-Methyljasmonate is preferably converted. •  The enzyme specificity in hexane is similar compared to that in supercritical carbon dioxide.

29. Lipase-catalysed kinetic resolution of racemates at temperatures from 40°C to 160°C in supercritical CO2 Example: Phenylethanol The enzyme: Novozym 435, EC 3.1.1.3 from Candida antarctica B, 7000 PLU/g (activity expressed in propyl laurate units base on a batch synthesis assay), water content 1-2% w/w,

30. OH O O CH 2 C + C* CH CH 3 3 1-Phenylethanol (R,S) Vinylacetate O O O OH CH H C + 3 C* 3 CH 3 1-Phenylethylacetate Vinylalcohol Acetaldehyde Reaction Scheme

31. Reactive separation Carbon dioxide + r-1-Phenylethylacetate r,s-1-Phenylethanol s-1-Phenylethanol s-1-Phenylethylacetate Catalyst: Novozym 435 (Lipase) Vinylacetate Carbon dioxide, supercritical s-1-Phenylethanol Carbon dioxide

32. Structure of the enzyme Lipase Arctica candida

33. Separation of r,s-1-phenylethanol Yield rel. To racemate [%] Reaction time [h] Conversion of (R,S)-1-phenylethanol at 95°C () and 136°C () for phenylethanol esterification at 15 MPa.

34. Effect of temperature on reaction rate 1-Phenylethanol - vinylacetate - CO2 Reference: 1g immob. Enzyme, 100 oC Novozym 435 Initial substrate concentration: 0,5 M

35. Comparison of reaction rate

36. Pressure dependence Yield racemate conversion mmol mg-1 h-1 Pressure [MPa] Pressure dependence of the enzyme activity at 60°C of 1-phenylethanol reaction.

37. Enantiomeric excess Enantiomeric excess ee [%] of 1-phenylethanol () and ibuprofen () reaction respectively enantiomeric ratio E of ibuprofen ().

38. Reaction in n-hexane Yield of the reaction of 1-phenylethanol with vinyl acetate in n-hexane in dependence on substrate quantity. Solvent: 4 ml

39. Reaction in n-hexane Reacted quantity Substrate quantity Initial reaction rate in dependence on substrate quantity. Non linear fit to Michaelis-Menten eq.

40. Reaction in n-hexane Eadie-Hofstee-diagram

41. Reactor types CO2 Substrate Fixed bed Cycle pump CO2 Fixed bed tubular reactor Stirred tank reactor

42. Reaction in n-hexane tubular fixed bed reactor stirred tank reactor Comparison of stirred tank and tubular fixed bed reactor

43. Flow scheme of batch apparatus 1: CO2-feed, 2: CO2-cooler, 3: CO2 -pump, 4: inlet valve, 5: outlet valve, 6: spindle press, 7: reactor valve, 8: reactor pressure gauge, 9: injection valve, 10: view cell, 11: sample valve, 12: stirrer

44. Yield Ibuprofen Esterification in sc-CO2 Yield Time

45. Enantiomeric excess (ee) vs yield Yield

46. Temperature dependence of reaction rate

47. Temperature dependence of enantioselectivity

48. Esterification of FAEE Novozym 435 in 2-propanol

49. Transesterification with glycerol in n-hexane Enzyme: Lipase Novozym 435

50. Transesterification with glycerol in n-hexane (no enzyme)