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 Louisiana Tech University, Ruston, LA 71272 Ming Tien, Penn State University, University Park, PA 16802

Outline Introduction Catalytic Cycle of Peroxidases 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

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

Structure of Iron-Protoporphyrin IX Fe N N COOH COOH

Mn-Peroxidase (P. chrysosporium)

Representative Structure of Lignin Adapted from Adler

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

Catalytic Cyle of Peroxidases

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

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

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).

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

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

QCM Characterization of Nano-assembly on a Flat Surface Film Thickness is calculated using Sauerbrey equation: ΔT (nm) ≈ - (0.016 ± 0.002) x ΔF Δm (ng) ≈ - 0.87 x ΔF where, ΔF is frequency shift of QCM resonator after each layer is deposited 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

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

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

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

Effect of Polycations on Activities of Immobilized LiP

Effect of Number of Layers on LbL-Assembled MnP

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

Scheme for Oxidation of Substrates Reactant Product Active site Scheme for Oxidation of Substrates

Activity Assays of Assemblies on Flat surface: Effect of drying

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

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

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

QCM Characterization: With a composite layer of silica nanoparticles

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

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

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

VA Oxidation Using LiP and MnP on MF Microparticles

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

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.

Acknowledgement Louisiana Tech U – Start-up Grant

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

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

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

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 Δm (ng) ≈ - 0.87 x ΔF where, ΔF is frequency shift of QCM resonator after each layer is deposited 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