| Enzymes play a role in the production of pharmaceutical agents, fuels, alcoholic beverages, foods and other consumer products. In these production processes, the levels of carbon sources (e.g. glucose, maltose, starch) and their breakdown products can greatly affect the activity of the enzyme and quality of the desired end products. It is imperative to monitor these analytes in order to optimize manufacturing performance and attain maximum product yield.;In vitro microdialysis (MD) is a membrane-based sampling and sample clean-up technique. When coupled to reversed-phase high performance liquid chromatography (HPLC) or high performance anion-exchange chromatography (HPAEC), effective separation of carbohydrates can be accomplished. The direct, sensitive and selective determination of these sugars is achievable by pulsed electrochemical detection (PED), and in the case of chromophoric glycosides, UV absorbance detection. In this work, MD-HPAEC-PED and MD-HPLC-UV were used to quantitatively monitor saccharides and glycosides, respectively, in a variety of matrices.;Specifically, the model enzyme almond beta-glucosidase and phenolic glycoside substrates were used to investigate quantitative in vitro MD and to determine Michaelis-Menten enzyme kinetic parameters. In order to validate the method, Km and Vmax values obtained by MD-HPLC-UV were compared to standard spectrophotometric data and to literature values.;This method was extended to the determination of saccharides in laundry detergent wash cycles using MD-HPAEC-PED. Quantitative comparisons between thermostable amylases from the genus Bacillus were made based on of rate of starch breakdown and maltooligosaccharide formation, showing great advances over traditional colorimetric methods. Lastly, the MD-HPAEC-PED method was optimized to monitor glucoamylase hydrolysis of liquefied corn mash, an important step in fuel ethanol production. In addition to reduced sample preparation, these instrumental systems have applications for the direct, on-line monitoring of a broad range of substrates and products in diverse enzymatic processes. |
