Download Jon Magnuson, Glenn Fryxell, Linda Lasure, Doug Elliot (PNNL)

Immobilized Enzymes in Functionalized
Nanoporous Materials Exhibit Enhanced
Activity and Stability
Eric J. Ackerman, Chenghong Lei,Yongsoon Shin (PNNL)
Jon Magnuson, Glenn Fryxell, Linda Lasure, Doug Elliot
(PNNL)
Jun Liu (PNNL, now at Sandia)
Hydrolysis of Organophosphorus by Immobilized OPH
Stable enzymes
entrapped in nanopores
may one day be
routinely used for
chemical reactions.
Enzymes in this
environment are stable
for extended periods of
time.
J. Am. Chem. Soc. 2002,
124, 11242−3
Potential Applications
Enzymes are nano-machines of cells, catalyzing thousands of useful chemical reactions.
Microscopic reversibility means that outside cells, reactions A --> B and B --> A are feasible.
Unlike typical chemical catalysts, enzymatic reactions occur at ambient conditions; i.e. green
technology.
Enzyme fragility has been a primary limiting factor in applications.
Our breakthrough is applicable at multiple scales: sensors to industrial reactions
Focus areas:
homeland security
energy
Why is this a breakthrough?
Decades of work immobilizing enzymes has yielded small amounts of mostly inactive
enzyme.
Previous approaches generally destroyed the enzymes activity as a consequence of the
immobilization procedure. This occurred either by killing the enzyme or burying it inside
a material so that substrates and products could not enter and leave.
Specific activity (enzyme activity per amount of enzyme) is the important parameter.
We immobilize larger quantities of active enzyme per amount of material than other
methods.
Our immobilized enzyme exhibits enhanced stability and, for the first time, enhanced
activity.
Maintaining and Promoting Enzyme Activity
Confinement can eliminate some expanded configurations of the unfolded chain,
shifting the equilibrium from the unfolded state toward the native state.
Denatured Enzyme,
unfolded state in solution
Renatured Enzyme,
native state in a confined space
Biochemistry 2001, 40: 11289-11293.
Confined space: Mesoporous silica
60 nm
300 Å Mesoporous Silica
Confined space for enzyme (protein): Functionalized Mesoporous silica (FMS)
Si
CH2
CH2
CH2
SH
Si CH2 CH2 CH2 NH2
Si CH2 CH2 COOH
Schematic drawing of FMS.
Feng, X.; Fryxell, G. E.; Wang, L. –Q.; Kim, A. Y.; Liu, J.; Kemner, K. M.
Science 1997, 276, 923-926.
OPH structural dimensions & amino acid residues
92 Å
40 Å
56 Å
OPH structure with charged surface residues: lysine (red), arginine (green),
glutamic acid (yellow) displayed by “ball and stick”. The majority of the protein is
displayed by “backbone”.
Covalently linking protein in FMS
O
Si
CH2
CH2
CH2
O
NH2 + H C CH2
CH2
CH2
C H
O
Si
CH2
CH2
CH2
N
CH CH2
CH2
CH2
C H
O
Si
CH2
CH2
CH2
N
CH CH2
CH2
CH2
C H + H2N
Si
CH2
CH2
CH2
N
CH CH2
CH2
CH2
CH
N
Protein
Protein
Reaction of NH2-FMS with GDAH and subsequently with the enzyme.
Spontaneously entrapping protein in FMS
Quic kTime™ and a Sorenson Video 3 dec ompress or are needed to see this picture.
Organophosphorous Hydrolase (OPH)
Structure of Organophosphorus Compounds:
Toxicities of Organophosphorus compounds:
O
CH3
(CH3)2CH O P F
Soman
O
CH3CH2 O P SCH2CH2N
CH3
CH(CH3)2
O
CH2CH3
Paraoxon
150-600
Parathion
13
Paraoxon
0.5
Sarin
0.01
Soman
0.01
Tabun
0.01
VX
0.001
Palytoxin
0.00015
Botulinum toxin
0.000001
CH(CH3)2
VX
O
Diazinon
CH3
Sarin
CH3CH2 O P O
Approx. LD50
(mg/kg, iv.)
O
(CH3)3C CH O P F
CH3
Compounds
NO2
O
(CH3)2N P CN
O CH2CH3
Tabun
Comparison of different porous silica support for OPH immobilization
250
OPH Protein Amount
0.05
OPH Activity
0.04
200
150
0.03
100
0.02
50
0.01
0
5000
OPH Entrapped in HOOC-FMS
OPH Specific Activity
(Units/mg of protein)
2%
N
PS
H
20 O U
% O MS
H C
2% O -NP
O
S
20 HO C-N
% O P
H C- S
O FM
2 % OC S
-F
20 NH MS
% 2N N
2% H PS
220 NH NP
% 2- S
N FM
H
2- S
FM
S
0
OPH Activity
(units/mg of support)
OPH Protein Amount
(mg/mg of support)
0.06
4000
Free OPH in solution
3000
2000
1000
0
0
36
54
Time (days)
Enhanced Specific Activity & Stability of Immobilized OPH
145
Biosensin, Filtation, Decontamination
Paraoxon
O
CH3 CH2
O
P
O
O
CH2 CH3
NO2
OPH
H2 O
O
CH3 CH2
O
P
OH
+
HO
NO2
O
CH2 CH3
p-nitrophenol
Electrochemical Biosensing of Immobilized OPH in FMS
to Organophosphorus
0.90x10-4
Buffer
flush
i /A
Paraoxon addition
0.70x10-4
0.50x10-4
0.30x10-4
0
100
200
300
t/s
At 0.90V.
400
500
What’s next?
(1) We will integrate our extensive experiments with modeling/computation approaches
To understand how enzyme stability and catalytic activity are enhanced;
To better design nanomaterials;
To screen the desired enzymes by genetic engineering;
(2) We will try other enzymes of strategic significance, such as hydrogenase;
(3) We will also try alternative nanomaterials, especially conductive one
instead of silica;
(4) Design and fabrication of biosensing devices and filtration/decontamination
systems for Homeland Security, Army, and Environmental Protection.