Figure 1. Recombinant expression of OPH
Eric Ackerman
Enzymes are known to catalyze more than 5000 diverse chemical reactions. Enzymatic reactions occur at ambient temperatures and pressures, while chemical catalysts frequently require robust reaction vessels containing dangerous solvents maintained at extremely high temperatures and pressures. Accordingly, the simplified reaction conditions of enzymatic reactors require less complex engineering than catalytic reactors. Our focus is to develop the capability to build enzymatic micro-reactors. Our initial choice in enzymes was selected. Production of a successful single channel reactor represents a first step toward creating machines that mimic complex, multi-step chemical reaction pathways.
OPH is an attractive proof-of-principle enzyme for our first functioning micro-reactor because it catalyzes a commercially and nationally useful reaction; thus this project is not merely an academic exercise. The three-dimensional strructure and catalytic mechanism of OPH is known, thereby providing invaluable information for linkage to a suitable surface. Nonetheless, it is useful to begin considering other useful enzymes for future micro-reactors, especially if these enzymes are useful in new technologies such as applications in space exploration or carbon management.
The necessary task to produce active enzyme via recombinant DNA technology has already been accomplished. It is essential to use recombinant enzymes for this proposal if enzyme modification is necessary for attachment to suitable surfaces. Sufficient quantities of OPH have been achieved for one version of the enzyme. The most difficult part of the projectl is to covalently link OPH to a suitable surface without destroying its enzymatic activity. If our current OPH requires modification, then we must produce a new version of OPH. In a worst case scenario, there may 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 micro-reactors. The final task will require participation by the engineering members of the Nanotechnology Initiative.
Figures 1 and 2 demonstrate that we have succeeded to:
(1) engineer the OPH gene into an expression vector
(2) find conditions for expressing large quantities of OPH protein in bacteria
(3) purify OPH
Figure 1. Recombinant expression of OPH
Figure 2. Purification of OPH
Both figures show SDS-polyacrylamide gels. The first figure shows two different versions of OPH. The larger OPH species contains an extra 20 amino acids at its amino-terminus that facilitate purification, and may also enable attachment of purified OPH to a nano-surface. Both OPH species are clearly the predominant bands in their lanes, thereby indicating that the bacteria are expressing large quantities of OPH. Two different clones of OPH without the extra 20 amino acids are shown (lanes 3-4) and six different clones of OPH with the extra 20 amino acids are shown (lanes 5-10). As expected, the bacteria do not express OPH unless they are induced (compare lane 2 (no induction) with lanes 3-10 (induction). The second figure shows that OPH can be substantially purified in one chromatographic step (compare purity of the starting material in lane 2 with the eluted fractions in lanes 3-8).
Lilia K. Koriazova (AWU postdoctoral fellow) performed much of the work in the project.
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