nano-assembly of immobilized enzymes for biocatalysis in aqueous and non-aqueous media
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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
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