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Presented by Curtis Walton Supervisor Dr. Roberto Chica November 24, 2011

Carrier-Free Enzyme Immobilization for Stabilization of Biocatalysts. Presented by Curtis Walton Supervisor Dr. Roberto Chica November 24, 2011. Introduction. Enzymes are the most powerful catalysts: High catalytic activity Rate enhancements of 10 7 -10 17 High turnover number

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Presented by Curtis Walton Supervisor Dr. Roberto Chica November 24, 2011

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  1. Carrier-Free Enzyme Immobilization for Stabilization of Biocatalysts. Presented by Curtis WaltonSupervisor Dr. Roberto ChicaNovember 24, 2011

  2. Introduction Enzymes are the most powerful catalysts: • High catalytic activity • Rate enhancements of 107-1017 • High turnover number • High selectivity • Stereo-, chemo-, and regioselectivity • Mild reaction conditions • Near-ambient temperature and pH • Environmentally Friendly • Biodegradable • Easy to produce Richard Wolfenden, Acc. Chem. Res. 2001, 34, 938-94 2

  3. Introduction Richard Wolfenden, Acc. Chem. Res. 2001, 34, 938-94 3

  4. Industrial Applications of Enzymes The catalytic power of enzymes allows for the production of many products on an industrial scale. However, general enzyme stability has limited enzymatic catalyst in industrial preparations. Bommarius, Biocatalysis, 2004, Wiley 4

  5. Enzyme Stability Denaturants disrupt the secondary structure of the enzymes resulting in catalytically inactive enzymes P. V. Iyer et al., ProcessBiochemistry 43 (2008) 1019-1032 5

  6. Overcoming Enzyme Stability Issues Several approaches have been used to overcome these deficiencies including: • Enzymes from extremophiles • pH extremes or high temperatures • Taq & Vent Polymerase • The enzymes may not exist in extremophiles Ksenia Egorova, Current Opinion in Microbiology Volume 8, Issue 6, December 2005, Pages 649-655 6

  7. Overcoming Enzyme Stability Issues • Genetic manipulation of enzymes by rational design and directed evolution Genetic manipulation to stabilize enzymes can be a time consuming process requiring multiple rounds of screening mutants, especially to gain large in enzyme stability beyond the native enzyme Bertus Van den Burg et al., PNAS March 3, 1998 vol. 95 no. 5 2056-2060 Christopher K. Savile, et al. Science 329, 305 (2010); 7

  8. Enzyme Immobilization • Immobilization is the most widely used technique. This is due to: • Ease of separation of the enzyme from the reaction products • Enzyme reuse is economically advantageous (reduction in waste and catalyst production) • Stabilization of the enzyme 8

  9. Overcoming Enzyme Stability Issues • Immobilization Immobilization provides a fast method for stabilizing enzymes by restricting the enzymes structural mobility R. A. Sheldon, Adv. Synth. Catal. 2007, 349, 1289-1307 P. Lopez-Serrano et al., BiotechnologyLetters 24: 1379-1383, 2002 9

  10. Enzyme Immobilization • There are two approaches for immobilizing enzymes: • Carrier-bound immobilization • Carrier-free immobilization P. Lopez-Serrano et al., BiotechnologyLetters 24: 1379-1383, 2002 10

  11. Carrier-free immobilization Extensive research over the past 20 years has been performed for the immobilization of enzymes on carriers. However,carrier-free immobilization has several advantages: • Elimination of solid support carrier and reagents • Ease of preparation • Reduced activity loss • Increased space-time yield Carrier-free immobilization can be performed by cross-linking enzymes crystals and enzyme aggregates 11

  12. Cross-Linked Enzyme Crystals (CLECs) In the 1960’s cross-linking reagents were first used to stabilize enzyme crystals for crystallography. Richards et al., J. Mol. Biol. (1968) 37, 231-233 Guli, M., et al., AngewandteChemieVolume 122, Issue 3, pages 530-533, 10 DEC 2009 12

  13. CLECs- Glutaraldehyde • Glutaraldehyde’s first application as a cross-linking reagent was described by Quiocho and Richards in 1960’s. • It was determined to exist as monohydrates, dihydrates, cyclic hemi-acetals and oligomers. • The ratio of these forms of glutaraldehyde is dependent on pH, temperature and concentration. • The mechanism of protein reticulation by glutaraldehyde is poorly understood. Richards et al., J. Mol. Biol. (1968) 37, 231-233 H. Korn et al., J. Mol. Biol. (1972) 65, 525-529 M. Salem, et al., Acta Cryst. (2010). F66, 225-228 13

  14. CLECs - Glutaraldehyde Glutaraldehyde can exist in several forms that could react with lysine residues. Richards et al., J. Mol. Biol. (1968) 37, 231-233 H. Korn et al., J. Mol. Biol. (1972) 65, 525-529 14

  15. CLECs - Glutaraldehyde • A proposed mechanism for the reaction between glutaraldehyde and proteins Cross-linking by glutaraldehyde could occur through schiff base formation but the mechanism is still not fully elucidated. Thus, the optimal concentration of glutaraldehyde for cross-linking is determined empirically. Richards et al., J. Mol. Biol. (1968) 37, 231-233 H. Korn et al., J. Mol. Biol. (1972) 65, 525-529 15

  16. Determination of Optimal Glutaraldehyde Concentration Low concentrations of glutaraldehyde can result in the reconstitution of the soluble enzyme. Excess glutaraldehyde can restrict enzyme flexibility abolishing catalytic activity and disrupt the lattice structure. Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp. 7751-7754, 1994 16

  17. CLECs are Diffusion Controlled The crystallization procedure can be modified to form small enzyme crystals which is important to prevent mass-transfer limitations. Once the enzyme crystals are cross-linked determination of the catalytic and mechanical properties is necessary. A. L. Margolin, Angew. Chem. Int. Ed. 2001, 40, 2204-2222 17

  18. Analysis of CLECs To determine if a cross-linked enzyme crystal can be used as a biocatalyst several criteria must be evaluated. Can CLECs retain stability under hash conditions and for long periods of time? J. D. Vaghjiani et at., Biocatalysis and Biotransformation, Vol. 18, pp. 151-175, 1999 18

  19. Thermolysin CLECs • Thermolysin is a protease found in various Bacillus species. • Used in manufacturing of the artificial sweetener aspartame. 3N21 N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114, 7314-7316 19

  20. Preparation of Thermolysin CLECs • The purified enzyme was crystallized in 1M Calcium acetate, 30% dimethyl sulfoxide, 50 mMTris buffer, pH 7.0 • Cross-linked with 12.5% glutaraldehyde in 50 mMTris buffer for 1 hour and and subsequently lyophilized. • Rehydration of thermolysin CLECs allows for retention of 80% activity compared to soluble non-lyophilized enzyme. The prepared CLECs can be assayed for activity in the presence of organic solvents. N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114, 7314-7316 20

  21. Thermolysin CLECs on Organic Solvents • An equal volume of organic solvent was added to free and cross-linked enzyme preparations for 1 hour at 40oC • The enzyme activity was determined following the incubation period CLEC retain >95% activity in the presence of organic solvents. Can they retain activity at elevated temperatures? N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114, 7314-7316 21

  22. Thermolysin CLECs Stability • Thermolysin was incubated at 65oC for 5 days CLEC retain >90% activity during incubation at 65oC . CLECs retained activity at pH extremes. N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114, 7314-7316 22

  23. Thermolysin CLECs • Thermolysin was incubated with a mixture of proteases called Pronase CLECs are resistant to degradation by proteases. Can other enzymes be stabilized under these conditions? N. L. St. Clair et al., J. Am. Chem. Soc. 1992, 114, 7314-7316 23

  24. Aldolase CLECs • Rabbit Muscle Fructose diphosphatealdolase (RAMA) catalyzes a key carbon-carbon bond forming step in the synthesis of eukaryotic RNA polymerase inhibitor. • Due to its broad substrate specificity, RAMA has been exploited to synthesize other compounds. • However, many desired substrates are insoluble in aqueous medium. D-glyceraldehyde 3 -phosphate Dihydroxyacetone phosphate Can we apply CLEC technology to stabilize RAMA in organic solvents? Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp. 7751-7754, 1994 24

  25. Aldolase CLECs in Organic Solvents • An equal volume of organic solvent was added to free and cross-linked enzyme preparations for 1 hour at 40oC • The enzyme activity was determined following the incubation period CLECs remain active in thepresence of organic solvents. Are CLECs of RAMA stable for long periods of time? Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp. 7751-7754, 1994 25

  26. Aldolase CLECs CLEC RAMA 65% activity after 6 months High [10mg/mL] soluble RAMA and FDP Dilute solutions (0.2mg/mL) of soluble RAMA and FDP CLECs of RAMA remain active for over 6 months. Sobolov et al., Tetrahedron Letters, Vol. 35, No. 42, pp. 7751-7754, 1994 26

  27. CLECs Conclusions • CLECs retain enzymatic activity for significantly longer periods of time than the soluble enzyme • Thermolysin (>4 days) • Aldolase (>6 months) • CLECs retain enzymatic activity at higher temperatures (>40oC) than the soluble enzyme • CLECs retain enzymatic activity in the presence of organic solvents • However, the high cost of using purified enzymes crystals on industrial scale has limited their use 27

  28. Cross-Linked Enzyme Aggregates (CLEAs) • Proteins aggregate in the presence of high concentrations of salts. • Do aggregates retain activity? • Can we immobilize them in a similar fashion to CLECs? • How do we prepare enzyme aggregates? P. Lopez-Serrano et al., BiotechnologyLetters 24: 1379-1383, 2002 28

  29. Preparation of CLEAs • Purification and aggregation can be performed in a single step. However, are the aggregates active? Richard R. Burgess, Methods in Enzymology, Volume 463 29

  30. Activity of Penicillin Acylase CLEAs • Penicillin Acylase was the first enzyme to be tested for activity following aggregation • CLEA retain activity albeit lower than cross-linked crystals. What do these aggregates look like? Cao et al., Organic letters, 2000, Vol. 2, No. 10, 1362-1364 30

  31. Electron Micrograph of CLEAs • Unlike enzyme crystals, aggregates do not form a lattice but they do form a superstructure. An individual aggregate can millions of enzyme molecules. Since enzyme aggregates can be prepared very quickly can prepare a high throughput method to optimize the aggregates activity? R. Schoevaart, et al., Biotechnology and Bioengineering, Vol. 87, NO. 6, September 20, 2004 31

  32. High Throughput Development of CLEAs R. Schoevaart, et al., Biotechnology and Bioengineering, Vol. 87, NO. 6, September 20, 2004 32

  33. High Throughput Development of CLEAs • Not all precipitants are appropriate for use with each enzyme. It is necessary to determine the best precipitant for each enzyme to retain maximal activity. Some enzymes experience hyperactivation by precipitation. R. Schoevaart, et al., Biotechnology and Bioengineering, Vol. 87, NO. 6, September 20, 2004 33

  34. Hyperactivation of Lipase B CLEAs • Lipase B experiences a conformation change from a closed to open state resulting in hyperactivation of the active site that does not occur in the soluble enzyme. Do CLEA exhibit similar characteristics to CLEC in terms of stability. R. Schoevaart, et al., Biotechnology and Bioengineering, Vol. 87, NO. 6, September 20, 2004 U. Hanefeld et al., Chem. Soc. Rev., 2009, 38, 453–468 34

  35. Nitrile Hydratase CLEAs • >100,000 tons of acrylamide produced annually • Enzymatic process replaced heterogeneous copper catalyst at 120oC • The enzyme is unstable in cell free formulation and used in whole cell formulations • Product can be destroyed by cells (presence of amidases and protease make a cell-free methods preferable) S. Van Pelt et al, Green Chem. 2008, 10, 395-400 Brant J Bassam & Peter M Gresshoff Nature Protocols 2, 2649 - 2654 (2007)  35

  36. Preparation of Nitrile Hydratase CLEA with Glutaraldehyde • The optimal concentration of glutaraldehyde was determined by measuring the amount of remaining soluble enzyme. Does cross-linking increase stability of Nitrile Hydratases? S. Van Pelt et al, Green Chem. 2008, 10, 395-400 36

  37. Nitrile Hydratases CLEA stability • The nitrile hydratase is stable at 21oC for >21 days. Do CLEA of nitrile hydratase allow for stable production of acrylamide? S. Van Pelt et al, Green Chem. 2008, 10, 395-400 37

  38. Nitrile Hydratases CLEA Activity • The CLEA is capable of producing acrylamide and out performs whole cell and crude cell-free extracts. Can the CLEA be reused? S. Van Pelt et al, Green Chem. 2008, 10, 395-400 38

  39. Fed-batch production of Acylamide with CLEAs of Nitrile Hydratases • The CLEA is capable of producing acrylamide and out performs whole cell and crude cell-free extracts and were catalytically activity through multiple cycles. S. Van Pelt et al, Green Chem. 2008, 10, 395-400 39

  40. Antifouling CLEAs • To prevent fouling of vessels by microorganisms tributyltin was previously as an antifouling agent • However, tributyltin has been banned by European countries. • Protease have been shown to prevent fouling by degrading biofilms Each year fouling cost the shipping industry millions of dollars. • Are CLEAs capable of preventing fouling in salt water after being mixed and dried with marine paint? Messugeur et al., Progress in OrganicCoatings Volume 50, Issue 2, July 2004, Pages 75-104 Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629 J. Kim et al., Biotechnology Progress Volume 18, Issue 3, pages 551–555, 2002 40

  41. Subtilisin CLEAs in Artificial Salt Water • ASW – 547.6mM NaCl, 56.8 mM MgSO4x7H2O, 2.4 mMNaHCO3 • Incubated in Xylene for 24 hours • The CLEA remain activity in ASW and Xylene had no irreversible effect on the structure of the aggregates. Overall, the CLEA have a better stability in ASW although the unmodified subtilisin absolute rate of catalytic activity was highest. Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629 41

  42. Stability of CLEAsin dried marine paint • The free enzyme activity decrease over a 28 day period. CLEAs remain active over 28 days. Why does the free enzyme activity decrease significantly? Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629 42

  43. Hydration Layer of CLEAs in Marine Paint • Unmodified particles are extremely small (8 nm3) and will leach quickly from the paint resulting in a lost of activity, having aggregated enzymes is advantageous in reducing leaching of the enzymes. • As the hydration layer increase additional CLEA are exposed to ASW Skovgaard et al., J. Mater. Chem., 2010, 20, 7626-7629 43

  44. CLEA Conclusions • Simple method of generating immobilized enzymes. • Purification and aggregation in one step. • Increased storage lifetime compared to soluble enzyme. • Retain stability over multiple batch reactions • Retain stability in the presence of organic solvents 44

  45. Future Prospects • Renewable and selective catalytic technology • Increase in the number of enzymes used in industrial processes with CLEC/CLEAA technology such as biosensors • Potential for catalytic cascades (combination of multiple catalytically relevant enzymes aggregated together) 45

  46. Acknowledgements Thanks to Jamie Daveyand Samuel Oteng-Pabi. Thank you for listening! 46

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