Evaluation of the activity of beta-glucosidase immobilized on polydimethylsiloxane (PDMS) with a microfluidic flow injection analyzer with embedded optical fibers

摘要

beta-glucosidase from almonds was immobilized on a polydimethylsiloxane (PDMS) microdevice by covalent chain using 3-aminopropyltrietoxysilane and glutaraldehyde. Enzymatic activity was evaluated using p-nitro-phenyl-beta-D-glucopyranoside dissolved in a 0.01 M pH 5.0 phosphate solution at 45 degrees C measuring the reaction product (p-nitrophenol) at 410 nm. The microdevice consisted of two parts: the one part where the enzymatic reaction was carried out and a second part where pH was adjusted at 10, with NaOH. The reaction product was measured at the microchip exit using two optical fibers which were aligned facing each other with a gap of 7 mm, between both tips using guides located perpendicular to the flow outlet. A water bath was used to carry out the enzymatic reaction on the microdevice at 45 degrees C. The enzymatic surface of the PDMS microdevice was 1.15 cm(2) and the immobilized beta-glucosidase amount on the microdevice was of 1.17 g/cm(2). The calculated kinetics parameters were: Km 2.5 mM; Vmax 2.2 mM/min; Kcat 908.3/min and Kcat/Km 363.3/mM min. The immobilized enzyme is very stable decreasing only 5% the first 15 days; on the 30th day, the activity was 69%, regarding the initial activity.