PNNL-13501 - Pacific Northwest National Laboratory

• optimize attachment of organophosphorous hydrolase
(without destroying its enzymatic activity) to defined
surfaces containing high densities of enzyme-sized
pores
• assemble immobilized organophosphorous hydrolase
into improved microchannel reactors exhibiting high
throughput and durability
• identify the practical limits of an organophosphorous
hydrolase microchannel reactor in terms of
efficiency, flow rate, pressure drop, and durability
• test immobilized organophosphorous hydrolase in
traditional media and liquid foams to increase their
utility for multiple applications.
Results and Accomplishments
This project consisted of the following four tasks:
1. identification of future suitable enzymes besides
organophosphorous hydrolase
2. production of active enzyme via recombinant DNA
technology
3. attach enzymes to porous, nano-surface without
destroying enzymatic activity
4. assemble into a complete reactor and demonstrate
functionality.
We subcloned the organophosphorous hydrolase gene into
E. coli expression vectors and achieved ~2 to 4 g
organophosphorous hydrolase per liter (Figure 1). (This
is at least 100-fold higher expression than previously
reported and approaches the highest levels obtainable.)
We attached organophosphorous hydrolase to derivatized
silica containing enzyme-sized pores. Attachment
stoichiometries of ~50 to 100 mg organophosphorous
hydrolase per gram SAMMS retain 100% of enzyme
activity. Furthermore, immobilized organophosphorous
hydrolase better withstands treatment by acid, base, and
lyophilization than organophosphorous hydrolase free in
solution (Figure 2).
Figure 1. Sodium dodecylsulfate-polyacrylamide gel
demonstrating high organophosphorous hydrolase
expression (total E. coli lysates). Lane 1, molecular weight
markers; 2, pET11a-organophosphorous hydrolase before
induction; 3 and 4, pET11a- organophosphorous hydrolase
clones after induction; 5 through 10, pET15b-
organophosphorous hydrolase clones after induction.
OPH activity, %
100
The organophosphorous hydrolase-SAMMS material has
been fixed within a prototype microchannel reactor and
shown to have excellent throughput, catalytic ability, and
durability. Thus, our proof-of-principle experiments
established that a useful enzyme can 1) be produced
recombinantly at high yields, 2) be linked to suitable
materials containing enzyme-sized pores, 3) retain its
ability to inactivate a chemical weapon surrogate,
4) exhibit enhanced stability as a consequence of its
immobilization, and 5) be used within a high throughput
device that minimizes pressure drops but maximizes mass
transfer to the catalytic surface.
Acknowledgment
90
80
70
60
50
40
30
20
10
0
100
100
43.39
0h
1h
Time, hrs
We thank Jun Liu for making available his SAMMS
material for testing.
99.44
24h
1.55
81.9
OPH-SAMMS
Soluble OPH
Soluble OPH
OPH-SAMMS
Figure 2. Enhanced stability of organophosphorous
hydrolase immobilized to derivatized SAMMS.
Organophosphorous hydrolase covalently linked to SAMMS
and organophosphorous hydrolase at a comparable
concentration in solution was tested at pH 5.0 for 1 and 24
hours. Similar experiments demonstrate enhanced stability
to alkali treatment and lyophilization.
Materials Science and Technology 317