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NANO-ASSEMBLY OF IMMOBILIZED ENZYMES FOR BIOCATALYSIS IN AQUEOUS AND NON-AQUEOUS MEDIA

NANO-ASSEMBLY OF IMMOBILIZED ENZYMES FOR BIOCATALYSIS IN AQUEOUS AND NON-AQUEOUS MEDIA. Debasish Kuila, Ph.D. Professor and Chair of Chemistry North Carolina A&T State University Greensboro, NC 27411 dkuila@ncat.edu Yuri Lvov, Devendra Patel, Rajendra Aithal, and Gopal Krishna

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NANO-ASSEMBLY OF IMMOBILIZED ENZYMES FOR BIOCATALYSIS IN AQUEOUS AND NON-AQUEOUS MEDIA

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  1. NANO-ASSEMBLY OF IMMOBILIZED ENZYMES FOR BIOCATALYSIS IN AQUEOUS AND NON-AQUEOUS MEDIA Debasish Kuila, Ph.D. Professor and Chair of Chemistry North Carolina A&T State UniversityGreensboro, NC 27411 dkuila@ncat.edu Yuri Lvov, Devendra Patel, Rajendra Aithal, and Gopal Krishna Louisiana Tech University, Ruston, LA 71272 Ming Tien, Penn StateUniversity, University Park, PA 16802

  2. Outline • Introduction • Lignin Peroxidase (LiP) • Manganese Peroxidase (MnP) • Catalytic Cycle of Peroxidases • Layer-by-Layer Assemblies of LiP and MnP on a Flat Surface • Characterization using a Quartz Crystal Microbalance (QCM) • Using silica nanoparticles • Veratryl Alcohol Oxidation (aqueous and non- aqueous) • Nano-assemblies on Microparticles - Oxidation • Conclusions

  3. Lignin and Manganese Peroxidases Lignin Peroxidase Mn Peroxidase • Lignin Peroxidase • Heme access channel • Also site of long range transfer • Mn Peroxidase • Heme access channel • Mn binding site near heme

  4. Structure of Iron-Protoporphyrin IX N N Fe N N COOH COOH

  5. Mn-Peroxidase (P. chrysosporium)

  6. Representative Structure of Lignin Adapted from Adler

  7. Characteristics of LiP and MnP • Lignin Peroxidase (LiP) and Manganese Peroxidase (MnP) are isolated from Phanerochaete chrysosporium (Prof. Tien, Penn State). • LiP: Molecular Weight ~42,000, PI ~3.5 – 4.0 • MnP: Molecular Weight ~45,000, PI ~4.5 • Oxidize aromatic substrates of higher redox potential – a distinct feature

  8. Catalytic Cyle of Peroxidases

  9. Oxidation of an Alcohol by Ferri-LiP in the presence of H2O2 O O N N N N N N N N Fe(IV)+ ∙ Fe(IV)+ ∙ Fe(III) Fe(III) + H2O2 N N N N N N N N Ferric Enzyme Compound I R R H H C C + O H O - H2O H Ferric Enzyme Compound I Alcohol Aldehyde

  10. Why Do Immobilization of Enzymes? • Stabilize the enzyme… • Bioreactors • Oxidize Aromatic Pollutants • Bioremediation

  11. Enzyme Immobilization Procedure • Electrostatic interaction between oppositely charged species. • Polyelectrolytes: • Poly(dimethyldiallylammonium chloride) (PDDA) – PI ~13 • Poly(ethylenimine) (PEI) – PI ~11 • Poly(allylamine) (PAH) – PI ~ 8 • Poly(styrenesulfonate) (PSS) – PI ~2 • Enzymes: • Lignin Peroxidase (LiP) – PI ~3.5 • Manganese Peroxidase (MnP) – PI ~4.5 • LbL assembly carried out at pH 6.0 (Acetate Buffer).

  12. N+ H2 NH3+ Cl- Cl- N H3C CH3 SO3 - Na+ Structure of Polyelectrolytes PEI Poly(ethyleneamine) PAH Poly(allylamine) PDDA Poly(dimethyldiallylammonium) PSS Polystyrenesulfonate

  13. Initially Negatively Charged Surface Adsorption of Polyanions Adsorption of Polycations + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Polycation Polyanion Protein Adsorption of Polycations Adsorption of Protein + + + + + + + + + + + + + + + + + + + + + + + + + + + + LbL Assembly on a Flat Surface

  14. QCM Characterization of Nano-assembly on a Flat Surface Film Thickness is calculated using Sauerbrey equation: ΔT (nm) ≈ - (0.016 ± 0.002) x ΔF where ΔF is frequency shift of QCM resonator after each layer is deposited

  15. Effect of not drying enzyme layers (on thickness) Presence of water is critical for nano-assembly.

  16. Atomic Force Microscopy (AFM) Picture of (PDDA/MnP) Assembly on mica

  17. CH2OH CHO H2O2 OCH3 OCH3 OCH3 OCH3 Veratryl Alcohol Activity Studies of LbL-assembled LiP and MnP Veratryl Aldehyde (310 nm)

  18. Effect of Polycations on Activities of Immobilized LiP

  19. Effect of Number of Layers on LbL-Assembled MnP

  20. Effect of Number of Runs on Activity of (LiP/PEI)6 Nano-Assembly

  21. Reactant Product Active site Scheme for Oxidation of Substrates

  22. Activity Assays of Assemblies on Flat surface: Effect of drying

  23. Effect of acetone on Veratryl Alcohol Oxidation using (MnP/PEI)7 Assembly D. S. Patel et al, Colloids & Surfaces B: Biointerfaces,2005, 43, 13-19

  24. CH2OH CHO H2O2 OCH3 OCH3 OCH3 OCH3 Veratryl Alcohol Veratryl Aldehyde (310 nm) Effect of acetone on VA Oxidation using (MnP/PEI)7 Assembly • Colloids & Surfaces B: Biointerfaces, 2005, 43, 13-19

  25. Silica Nanoparticle (45nm) Protein Polyanion Polycation Polyanion Adsorption Positively Charged MF Particle (5 microns) Polycation Adsorption Protein Adsorption Assembly on Colloidal Particles Assembly on flat surface using a composite layer of silica nanoparticles

  26. QCM Characterization: With a composite layer of silica nanoparticles

  27. Effect of a composite layer of silica on activities of LbL-MnP

  28. Silica Nanoparticle (45nm) Protein Polyanion Polycation Polyanion Adsorption Positively Charged MF Particle (5 microns) Polycation Adsorption Protein Adsorption Assembly on Colloidal Particles Assembly on flat surface using a composite layer of silica nanoparticles

  29. Zeta Potential - MnP Assembly on Melamine Formaldehyde (MF, 5 microns)

  30. VA Oxidation Using LiP and MnP on MF Microparticles

  31. 2,6-Dimethoxyphenol Oxidation Using LiP/MnP on MF Microparticles Oxidation of 2,6-dimethoxyphenol

  32. Conclusions • Nano-Assemblies of LiP and MnP are successfully fabricated and characterized on a flat surface as well as colloidal particles. • A unique dynamic adsorption-desorption of enzyme layer during assembly process is observed using QCM. • Time, number of runs, non-aqueous media, and drying of the enzyme layers have significant effect on the activity of the LbL assembled enzymes. • A novel concept of using of silica nanoparticles improves bio-catalysis. • Oxidations of veratryl alcohol and 2,6 – dimethoxyphenol by enzymatic nano-assemblies on MF particles have been successfully demonstrated.

  33. Acknowledgement • Louisiana Tech U – Start-up Grant

  34. VA Oxidation in aqueous and aq-acetone media with MnP-PAH (4 layers) [Reverse Process]

  35. Colloids & Surfaces B: Biointerfaces, 2005, 43, 13-19 .

  36. Effect of Time on Activity of LbL Assembled Enzymes [ (MnP/PEI)5 ]

  37. Characterization of MnP-Assembly with Different Polyelectrolytes on a Flat Surface Using QCM Film Thickness is calculated using Sauerbrey equation: ΔT (nm) ≈ - (0.016 ± 0.002) x ΔF where ΔF is frequency shift of QCM resonator after each layer is deposited D. S. Patel et al, Colloids & Surfaces B: Biointerfaces,2005, 43, 13-19

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