Development of Sensing Systems for Dissolved Carbon Monoxide Concentration Using a Mo–Cu Dependent Carbon Monoxide Dehydrogenase

Abstract

Carbon monoxide (CO) generated in notable amounts from industrial processes as well as gasification of carbon-containing compounds is widely used as feedstock in biological and chemical gas conversion to value-added platform chemicals. In a typical CO conversion process, CO is supplied in gaseous form and the CO in dissolved form (liquid-phase) can be subsequently converted into valuable products including alcohols, hydrocarbons, and organic acids via biological or chemical catalysis. For optimal performance of this CO conversion process, monitoring dissolved CO concentration, Cco is therefore integral because Cco determines the gas-liquid mass transfer coefficient, a key parameter for the biological and chemical gas conversion. CO in gaseous form can be directly determined by gas-chromatography. However, CO is sparingly soluble in water, making the dissolved CO concentration more challenging to determine. Typically, Cco is indirectly estimated by measuring the concentration of CO in the gas phase and then Cco is calculated employing Henry’s law (assuming thermodynamic equilibrium). Therefore, this indirect method is limited because the Cco during most biological and chemical CO conversions are dynamically changing, and often a single measurement and complete analysis generally take over 10 minutes. The work presented in this thesis was motivated by the need for a direct determination of Cco. The core of this thesis is focusing on exploiting the intrinsic specificity of enzymes capable of catalyzing CO reaction as the biorecognition element in an enzyme-based biosensor. The first element of the work described in this thesis is the selection of the enzyme that preserves biocatalytic CO oxidation activity upon immobilization on the electrode surface. Next, heterologous expression of the target gene encoding the enzyme Mo–Cu dependent carbon monoxide dehydrogenase (CODH) in E. coli was secured for the convenient production of enzymes to be used in the development of the enzyme-based biosensor. With the expression system secured in hand, two different architectures sensing systems for monitoring Cco were explored. The first design is the use of recombinant Mo–Cu CODH as the CO-catalyst unit (CO to CO2) in front of a CO2 transducer. The developed CO-biomicrosensor exhibited approximately 95% sensor response towards CO in 5 min with linear sensor response towards quite low CO concentrations (0–9 µM) and a LOD of 15 nM. The sensor was approximately retained activity and sensitivity of 80% for one-week continuous operation. The second architecture is the enzyme immobilized on the electrode surface, in which the electrons generated from the CO oxidation to CO2 by CODH are transferred and collected at the electrode surface can be quantified as current. The main challenge of this second enzyme biosensor architecture is establishing stable directed immobilization that retains the biocatalytic function of the enzyme and secured efficient electrical connection between the active site and the electrode surface. To address this challenge, genetic modification of Mo–Cu CODH to fuse solid-binding peptides at a specific site was attempted. The “wire-ready” synthetic CODH-Ls demonstrated bi-functional activities, of CO oxidation, and solid-binding activity, as confirmed via conventional enzyme solution assay, atomic force microscopy (AFM), and quartz crystal microbalance (QCM), respectively. The next research question that was investigated in this study is the effect of the distance between immobilization point(s) and the enzyme active site on the electron transfer kinetics at the enzyme-electrode interface. To address this specific question, three synthetic CODH-Ls having gbp fused at N- or/and C-terminus were specifically designed to control immobilization of the enzyme in different points or site, and therefore the distance between the active site and the immobilization point varies accordingly, hence resulting in different electron transfer kinetics at the enzyme-electrode interface. The electrochemical result confirmed that only gbp(N)-CODH-L-gbp(C) is capable of direct electron transfer because the presence of the gbp at both N- and C-terminus facilitate a shorter distance between the enzyme active site and the electrode surface. The results exemplify the effect of the distance between enzyme immobilization point and active site to the electron transfer kinetics at the enzyme-electrode interface which is integral to the DET-based biosensor. Overall, the studies presented in this thesis outline the development of sensing systems for dissolved CO concentration using the enzyme, CODH as the CO catalyst unit. Taken together, the approaches demonstrated here is applicable not only for use in the development of CO sensing system, but also other application not limited to the enzymatic-based biosensor, enzymatic fuel cell, enzymatic electrosynthesis which can be tailored by using different enzyme, protein, or other biomolecules.