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 KrishnaLouisiana Tech University, Ruston, LA 71272

Ming Tien, Penn State University, University Park, PA 16802

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

Lignin Peroxidase•Heme access channel•Also site of long range transfer

Mn Peroxidase•Heme access channel•Mn binding site near heme

Lignin Peroxidase Mn Peroxidase

Lignin and Manganese Peroxidases

Fe

N

N N

N

COOHCOOH

Structure of Iron-Protoporphyrin IX

Mn-Peroxidase (P. chrysosporium)

C

C

16

15

14

13

11

10

9

8

7

65

4

3

2

112

O

CH

CH

CH2OH

H3CO

O

COH

CH

CH2OH

H3CO

OH

CH

H3CO

CHOH

HC

HOCH2

H3CO

O

O

HC

CH

CH2OH

HC

HC

OCH3

HOH2C

OCH3

OO

H3CO

O

CH

CH2OH

OCH3

O

CHOH

CH

CH2OH

OCH3

OH

CH

HC

CH2OH

H3CO OCH3

O etc.

O

C

C

C

H3CO OCH3

Carbohydrate

OH

C

HC

CH2OH

OCH3

O

O

CHOH

HC

HOH2C

OCH3

CHOH

CH

CH2OH

O

HC

H3CO

HO

OCH3

O

HC

HC

H2C

OCH3

OCH2

O

OCH

CH

O

CH2OH

H3CO

HC

CH2OH

OHOCH3

CH CH2OH

O etc.

Representative Structure of LigninRepresentative 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 PeroxidasesCatalytic Cyle of Peroxidases

Fe3+

+ H2O2 Fe4+O

+ H2O+.Ferric Compound I

Fe4+

O+.

+ RH Fe4+

O

+ R.Compound I Compound I I

Fe4+

O

+ RH Fe3+ + R.

Compound I I Ferric

Fe(III)

N N

N N

+ H2O2

O

C

H

H

H

Fe(III)

N N

N N

+O

C

H

- H2O

Ferric Enzyme Compound I

Compound I Ferric EnzymeAlcohol Aldehyde

Fe(IV)+ ∙

N N

N N

O

Fe(IV)+ ∙

N N

N N

O RR

Oxidation of an Alcohol by Ferri-LiP in the presence of H2O2

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).

N

CH3H3C

Cl-

PEIPoly(ethyleneami

ne)

PAHPoly(allylamine)

N+

H2

Cl-NH3+

PDDAPoly(dimethyldiallylammonium)

SO3 -

Na+

Structure of Polyelectrolytes

PSS

Polystyrenesulfonate

LbL Assembly on a Flat Surface

+++++++

+++++++

Initially Negatively Charged Surface

Adsorption of Polycations

Adsorption of Polyanions

Adsorption of Polycations

Adsorption of Protein

Polycation

Polyanion

Protein

+++++++

+++++++

+++++++

+++++++

+++++++

+++++++

+++++++

+++++++

+++++++

+++++++

QCM Characterization of Nano-assembly on a Flat Surface

0

200

400

600

800

1000

1200

1400

Null

PDDA/PEI/P

AHPSS

PDDA/PEI/P

AHPSS

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

Layers

Fre

qu

ency

Sh

ift

(Hz

)

02468101214161820

Film

Th

ickn

ess

(nm

)

MnP/PDDA

MnP/PEI

MnP/PAH

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)

-500

0

500

1000

1500

2000

Null PEI PSS PEI PSS PEI PEI PEI PEI PEI PEI PEI

Layers

Fre

qu

ency

Sh

ift

(Hz)

-5

0

5

10

15

20

25

30

35

Film

Th

ickn

ess

(nm

)

LiP/PEI

MnP/PEI

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

Veratryl Aldehyde (310 nm)

OCH3

CH2OH

OCH3

Veratryl Alcohol

OCH3

OCH3

CHO

H2O2

Effect of Polycations on Activities of Immobilized LiP

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

0 20 40 60 80 100 120

Time (min.)

Ab

sorb

ance

(31

0 n

m) (LiP/PDDA)5

(LiP/PEI)5

(LiP/PAH)5

Effect of Number of Layers on LbL-Assembled MnP

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 20 40 60 80 100 120

Time (min.)

Ab

sorb

ance

(31

0nm

)

(MnP/PAH) Layer = 1

(MnP/PAH) Layers = 2

(MnP/PAH) Layers = 4

(MnP/PAH) Layers = 7

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

0

0.05

0.1

0.15

0.2

0.25

0 20 40 60 80 100 120

Time (min.)

Ab

sorb

ance

(31

0nm

)

Assembly 1, Day 1, Run 1Assembly 1, Day 4, Run 2Assembly 2, Day 4, Run 1Assembly 2, Day 8, Run 2

Active site

Product

Reactant

Scheme for Oxidation of Substrates

Activity Assays of Assemblies on Flat surface: Effect of drying

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 20 40 60 80 100 120

Time (min.)

Ab

sorb

ance

(31

0 n

m)

(MnP/PEI)5, Drying of Enzyme LayerSkipped

(MnP/PEI)7, Drying Was Carried Outfor Characterization

Effect of acetone on Veratryl Alcohol Oxidation using (MnP/PEI)7 Assembly

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 20 40 60 80 100 120Time (min.)

Ab

sorb

ance

(31

0nm

)

Day 1, Aqueous

Day 2, 5% Acetone

Day 3, 10% Acetone

Day 4, 15 % Acetone

Day 5, 20 % Acetone

Day 6, 30% Acetone

Day 7, 35% Acetone

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

OCH

3Veratryl Alcohol Veratryl Aldehyde (310 nm)

OCH3

OCH3

CHO

H2O2

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 20 40 60 80 100 120

Time (min.)

Ab

sorb

an

ce (

31

0n

m)

Day 1, Aqueous

Day 2, 5% Acetone

Day 3, 10% Acetone

Day 4, 15 % Acetone

Day 5, 20 % Acetone

Day 6, 30% Acetone

Day 7, 35% Acetone

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

Assembly on Colloidal Particles

PolyanionPolycation

Positively ChargedMF Particle (5 microns)

Polyanion Adsorption

Polycation Adsorption

Protein Adsorption

Silica Nanoparticle (45nm)

Protein

Assembly on flat surface using a composite layer of silica nanoparticles

QCM Characterization: With a composite layer of silica nanoparticles

PDDA

PDDAPDDA

PDDA

PDDA

MnPPDDA

MnP

Null PDDAPSS PDDA

PSS

SILICA 45nm

PDDA

PSS

MnP

MnP

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

Null

PDDAPSS

PDDAPSS

PDDA

SILIC

A 45n

mPDDA

PSS

PDDAM

nP

PDDAM

nP

PDDAM

nP

PDDAM

nP

PDDA

Layers

Fre

qu

en

cy S

hif

t (H

z)

0

20

40

60

80

100

120

140

160

Film

Th

ick

ne

ss (

nm

)

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

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 20 40 60 80 100 120Time (min.)

Ab

sorb

ance

(31

0nm

) PDDA/Silica/(MnP/PDDA)4

(PDDA/MnP)7

Assembly on Colloidal Particles

PolyanionPolycation

Positively ChargedMF Particle (5 microns)

Polyanion Adsorption

Polycation Adsorption

Protein Adsorption

Silica Nanoparticle (45nm)

Protein

Assembly on flat surface using a composite layer of silica nanoparticles

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

MF

PSS

PDDA

PSS

PDDA

MnP 24 hrs

PEI PEI

MnP 24 hrs

MnP 24 hrs

-80

-60

-40

-20

0

20

40

60

80

Layers

Zet

a P

ote

nti

al (

mV

)

VA Oxidation Using LiP and MnP on MF Microparticles

0

0.05

0.1

0.15

0.2

0.25

0.3

0 20 40 60 80 100 120Time (min.)

Ab

sorb

an

ce (

310

nm

)

(PEI/LiP)2 on MF (5 microns)

(PEI/MnP)3 on MF (5 microns)

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

Oxidation of 2,6-dimethoxyphenol

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 10 20 30 40 50 60

Time (min.)

Ab

sorb

ance

(46

9 n

m)

(PEI/LiP)2 on MF (5microns)

(PEI/MnP)3 on MF (5microns)

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

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

0.2

0 2 4 6 8 10 12 14

Time (min)

Ab

so

rba

nc

e(3

10

nm

)

Aqueous media

5% acetone

10% acetone

15% acetone

20% acetone

25% acetone

AqueousR-same day

AqueousR-2nd day

AqueousR-3rd day

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

Comparisons

0

2

4

6

8

10

12

14

16

0 20 40 60 80 100 120

Time (min.)

Co

nce

ntr

atio

n (

nM

)

Native LiP ( Per ug of Enzyme)

(PEI/LiP)2 on MF (5 microns)

(PEI/LiP)5 on QCM

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

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60

Time (min)

Ab

sorb

ance

(31

0 n

m)

Day 1, Run 1

Day 3, Run 2

Day 6, Run 3

Day 21, Run 7

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

0

200

400

600

800

1000

1200

1400

Null

PDDA/PEI/P

AHPSS

PDDA/PEI/P

AHPSS

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

PDDA/PEI/P

AHM

nP

Layers

Fre

qu

en

cy S

hif

t (H

z)

0

2

4

6

8

10

12

14

16

18

20

Film

Th

ickn

ess (

nm

)

MnP/ PDDA

MnP/ PEI

MnP/ PAH

D. S. Patel et al, Colloids & Surfaces B: Biointerfaces, 2005, 43, 13-19