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 University Greensboro, NC
Yuri Lvov, Devendra Patel, Rajendra Aithal, and Gopal Krishna
Louisiana Tech University, Ruston, LA 71272
Ming Tien, Penn State University, University Park, PA 16802

2. 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

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 IXFe 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
Fe(III) N +
H2O2 O C H
- H2O
Ferric Enzyme
Compound I
Alcohol
Aldehyde
Fe(IV)+ ∙ R

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

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

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
Δm (ng) ≈ 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

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. Activity Studies of LbL-assembled LiP and MnP
OCH3
CH2OH
Veratryl Alcohol
CHO
H2O2
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. Scheme for Oxidation of Substrates
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. 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

25. 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

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

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

28. 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

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
Δm (ng) ≈ 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