Contact: David Brenchley
Pagemaster: Amanda Kissire
Updated: December 18, 2002
Eric J. Ackerman & Lilia K. Koriazova
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 is to develop the capability to build enzymatic microreactors; i.e., 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. In order to develop our capability with enzymatic reactors, our initial choice in enzymes is microbial organophosphorous hydrolase (OPH), which inactivates nerve gas as well as some pesticides. No additional enzymes or cofactors are required for this reaction. Producing a successful single channel reactor represents a first step toward creating machines that mimic complex, multi-step chemical reaction pathways.
This project consists of four tasks.
OPH is an attractive proof-of-principle enzyme for our first functioning microreactor because it catalyzes a commercially and nationally useful reaction. Once proof-of-principle is established, devices containing OPH microreactors could be constructed for insertion into individual gas masks, portable filtration units for vehicles, or permanent filters for buildings. These filtration units should efficiently inactivate nerve agents and protect their users. Similar devices could be constructed to inactivate pesticides.
The three-dimensional strructure and catalytic mechanism of OPH is known, thereby providing invaluable informa-tion for linkage to a suitable surface. Nonetheless, it is useful to begin considering other useful enzymes for future microreactors, especially if these enzymes are to be used in new technologies such as applications in space exploration or carbon management.
The second task, to produce active enzyme via recombinant DNA technology, has already been accomplished. It is essential to use recombinant enzymes for this project if enzyme modification is necessary for attachment to suitable surfaces. Sufficient quantities of OPH have been achieved for one version of the enzyme.
The third task is the most difficult part of the project because we must covalently link OPH to a suitable surface without destroying its enzymatic activity. If our current OPH requires modification, then we must repeat the second task to produce a new version of OPH. In a worst-case scenario, there may be several versions of OPH and dozens of surfaces and attachment chemistries that may require evaluation until success attachment is achieved. However, we may learn invaluable lessons from failed attachment methods and abandoned surfaces that will hasten future enzymatic microreactors.
The final task, assembling the device into a complete reactor and demonstrating its functionality, requires participation by staff of the Nanotechnology Initiative in a joint effort.
We succeeded in covalently linking wild-type OPH to an amino-derivatized SAMMS (self-asssembled monolayers on mesoporous silica) provided by Dr. Jun Liu (PNNL). Although this is a breakthrough because it demonstrates one successful attachment chemistry, this particular SAMMS was suboptimal for OPH because its pore size was too small for OPH to easily penetrate. We continue to test other surfaces and attachment chemistries. Interestingly, OPH immobilized to SAMMS showed enhanced stability compared to a similar concentration of OPH in solution. The enhanced stability is apparent with respect to pH and lyophilization.
We thank Jun Liu for making his SAMMS material available for our testing purposes.
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