Development of Isothermal Microcalorimetry with in-situ Electrochemical Enzyme Sensors: A Novel Continuous Assay Technology for Kinetic Analysis of Enzymatic Hydrolysis of Cellulose

Nicolaj Cruys-Bagger

Studenteropgave: Speciale

Abstrakt

In this work we present the development of a novel combination of isothermal microcalorimetry and electrochemical enzyme sensors with special reference to kinetic analysis of enzymatic hydrolysis of lignocellulosic biomass suspensions. The work was motivated from that one of the key challenges in enzymatic biomass research lies in the limited selection of simple, fast and quantitative assays for cellulolytic enzymes activity. Isothermal microcalorimetry has recently shown promise as a novel real-time assay based on quantification of the heat associated with the enzymatic degradation of the biomass. To improve the specificity in the calorimetric signal we modified a Thermometric TAM2227 isothermal microcalorimetric titration vessel so it could be equipped with dual miniaturized working electrochemical sensors, reference- and counter electrode in a bi-potentiostat setup. This setup allows for different measurement opportunities of either two different types of sensors working in concert or interference detection by a ‘compensating’ electrode. Miniaturized amperometric glucose sensors were constructed in glass capillary tubes, outer diameter 1 mm, by membrane entrapment of glucose oxidase that was immobilized on the surface of a carbon paste electrode with a mixed mediator, p-benzoquinone. An oxidation current of the reduced redox mediator, by the glucose oxidase reaction, was recorded at a fixed potential of +0.6 V vs. Ag/AgCl. The oxidation current thus obtained was proportional to the concentration of glucose up to 25 mM. We investigated the performance of this novel combined technique in terms of advantages and limitations. It was found that the miniaturized amperometric glucose sensors could be incorporated into the calorimetric vessel without causing any significant interference in the calorimetric baseline signal from air leaks and any significant consumption of glucose. Optimization of the combined experimental setup is still needed but the combined method shows promise for continuous in-situ monitoring of glucose and simultaneous heat detection. The method was planned to be used to study the enzymatic hydrolysis kinetics of crystalline cellulose by cellulolytic enzymes. Where the calorimetric signal scales with the overall activity of the all cellulolytic enzymes in the mixture, then the glucose sensor detects only the activity of glucose producing enzymes. It would thus be possible with the combined method to study the enzyme kinetics in more detail than otherwise possible with the calorimetric assay alone. This gives a unique opportunity for a deeper insight into the synergetic effect of cellulolytic enzymes working in concert. Hence, it was concluded that the novel combination technique of isothermal microcalorimetry and electrochemical enzyme sensors shows great promise as a powerful analytical tool, not only for characterization of cellulolytic enzyme mixtures activity in lignocellulosic biomass degradation, but also as an general application of electrochemical enzyme sensors as specific sensors in calorimetric instruments for improved specificity. In comparison with conventional assay methods, based on chromatography or spectrophotometry, the presented method has the advantages of being free from the influence of the optical properties (turbidity and coloration) of the reaction mixture, and provides monitoring in real-time. The electrochemical glucose sensor was utilized to study the enzymatic hydrolysis kinetics of microcrystalline cellulose, suspended in acetate buffer (pH 5), by a commercial cellulase mixture. The result shows that electrochemical glucose sensor has big potential as an independent assay to study the kinetics of enzymatic hydrolysis of cellulose substrates.

UddannelserFysik, (Bachelor/kandidatuddannelse) KandidatKemi, (Bachelor/kandidatuddannelse) Kandidat
SprogEngelsk
Udgivelsesdato16 jul. 2010
VejlederePeter Westh, Tage Emil Christensen, Hirosuke Tatsumi & Kim Borch

Emneord

  • calorimetry
  • electrochemical enzyme electrodes
  • enzymatic hydrolysis