PNNL-13501 - Pacific Northwest National Laboratory

Study Control Number : PN98028/1274
Enzymatically Active Surfaces
Eric J. Ackerman
Immobilized enzymes applied to microchannel applications represent a potential improvement in combining proteins with
inorganic materials to create devices and materials with exciting new properties and capabilities. Our long-term objective
is to use highly selective enzymes and protein ligands fused with micro- and nanotechnology to create microchannel
reactors and sensors.
Project Description
Enzymes are known to catalyze more than 5000 diverse
chemical reactions. Enzymatic reactions occur at ambient
temperatures and pressures, thereby obviating the need for
complex reaction vessels required by chemical catalysts.
Our focus has been to develop the capability to build
enzymatic microreactors and to demonstrate catalytic
activity (as an example of enhanced microsystem
performance through highly functional surfaces in
microchannels). Building enzymatic microreactors
requires 1) producing active enzymes, 2) linking the
enzymes to suitable surfaces while retaining catalytic
activity, and 3) integrating the immobilized enzymatic
surface into a functional reactor. To develop our
capability with enzymatic reactors, our initial choice in
enzymes was microbial organophosphorous hydrolase,
which inactivates nerve gas as well as some pesticides.
No additional enzymes or cofactors are required for this
reaction. The goal was to produce recombinant enzyme,
immobilize it to a porous material without losing
enzymatic activity, test for enhanced enzymatic stability
as a consequence of immobilization, and assemble a
prototype microchannel reactor. We succeeded in
achieving all these goals, and production of a successful
single-channel reactor represents a first step toward
creating machines that mimic complex, multi-step
chemical reaction pathways.
Introduction
The combination of enzymes with micro- and
nanotechnology materials exploits the high specificity and
reaction speed (thousands of reactions per second) of
cellular nanomachines (enzymes) with the advantages of
high mass transfer and reaction kinetics possible only
through microchannel technology. Proteins outperform
traditional inorganic catalysts because they lower the
316 FY 2000 Laboratory Directed Research and Development Annual Report
energy barrier for a specific reaction, thereby reducing the
required conditions to ambient temperatures and
pressures. Recombinant DNA technology enables the
production of novel proteins with enhanced properties.
Microchannel enzyme bioreactors provide an innovative,
efficient technology to detect and/or destroy chemical and
biological weapons. Enzymes combined with
nanomaterials within microchannel devices are now
possible because of recent advances in materials sciences,
biotechnology, and microengineering. Thus, an
extremely high density of fully functional protein
becomes available to inactivate or bind substrate
efficiently (e.g., chemical agents and biological
pathogens) with high throughput.
The main drawback with enzymes and proteins is
fragility. Enzyme stability can be enhanced by
immobilization, but it is extremely difficult to immobilize
high densities of enzyme while retaining catalytic activity.
We have successfully developed methodologies to link an
enzyme that is important in detecting and destroying
chemical weapons to a nanomaterial. The enzyme,
organophosphorous hydrolase, was immobilized at a
density four times higher than previously known, and it
retained full enzymatic activity. Our enzymatic material
also exhibited markedly enhanced thermal and chemical
stability over the free enzyme. Moreover, we successfully
assembled the enzymatic material into a functioning
microreactor prototype that exhibits high throughput and
rapid reaction kinetics.
Approach
Our specific research objectives were to
• produce active recombinant organophosphorous
hydrolase, and if necessary, introduce sequence
changes to facilitate attachment to defined surfaces