Paper BI-FrM3
Reversible Biofunctionalization and Catalytic Activity of a Metabolic Pathway Enzyme in Reusable BioMEMS Devices

Friday, October 19, 2007, 8:40 am, Room 609

We report a reversible biofunctionalization strategy for assembling a biocatalytically-active enzyme at localized sites in reusable bioMEMS devices as a first step toward an experimental platform for metabolic engineering applications, e.g. drug discovery. We prefabricate an integrated bioMEMS device and exploit a non-permanent sealing and package design which supports programmable bio-component assembly at selected sites in the completely packaged microfluidic environment. The aminopolysaccharide chitosan is utilized as the interfacial biofunctionalization material for (1) the chemical signal-guided conjugation of chitosan to a Pfs enzyme through its pro-tag, which is genetically engineered at the C-terminal of Pfs and is activated by tyrosinase for the conjugation, and (2) the electric signal-guided electrodeposition of the Pfs-chitosan conjugate to a selective electrode under negative bias in the microfluidic channel. With biofunctionalizaton complete, the Pfs-mediated enzymatic reaction is performed by introducing the substrate S-adenosylhomocysteine (SAH) into the microchannel for conversion into the products S-ribosylhomocysteine (SRH) and adenine. Reaction solutions are collected and analyzed with high-performance-liquid-chromatography (HPLC). Initial studies show that high conversions are achieved at low flow rates, while much lower conversions are achieved at high flow. After reaction, a mild acid wash removes the assembled Pfs-chitosan conjugate and thus removes the catalytic activity. Subsequent biofunctionalization with Pfs-chitosan re-establishes enzymatic activity comparable to the original, illustrating the reversibility of the enzyme assembly and the reusability of our bioMEMS. Storage in PBS buffer at room temperature for 4 days degraded the conversion efficiency only slightly, demonstrating robustness of the assembled enzyme. This work demonstrates (a) the reversible assembly of a Pfs enzyme at a specific electrode address and (b) the efficacy of the metabolic pathway enzyme Pfs in the bioMEMS: Pfs converts SAH to SRH and adenine in one step of a multi-step cell-signaling process (autoinducer-2 production), a quorum sensing phenomenon that determines pathogenicity of bacteria. These results illustrate the capability of the bioMEMS as an efficient and reusable platform in screening potential enzyme inhibitors as antimicrobial drug candidates. This work is supported in part by the Robert W. Deutsch Foundation.