Investigation of micro-immobilised enzyme reactors containing endoglucanases for efficient hydrolysis of cellodextrins and cellulose derivatives
Publikation/Tidskrift/Serie: Analytica Chimica Acta
Förlag: Elsevier B V
Cellulases hydrolysing the interior parts of cellulose, also called endoglucanases, were immobilised in micro-immobilised enzyme reactors (RIMER) made of porous silicon with the purpose of investigating the use of such mu IMERs for hydrolysis of cellodextrins and soluble cellulose derivatives. The endoglucanases Trichoderma reesei Cel 12A (TrCel 12A) and Bacillus agaradhaerens Cel 5A (BaCel 5A) were covalently coupled to the surface of a silicon microchip through Schiff base formation. For characterisation cellohexaose was used as substrate for the immobilised enzymes. The characteristics of the RIMER were investigated by studying the product formation when varying the concentration, flow-rate, temperature and pH of the substrate solution. Hydrolysis was performed in the RIMER connected on-line to a chromatographic system, where the products were separated and detected using high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD). A comparison of the hydrolytic pattern between BaCel 5A and TrCel 12A was carried out and the results show that the two investigated endoglucanases give specific hydrolytic patterns in the products formed that provide important information about the enzymes. The RIMERs are robust and can be employed continuously over a period of at least several days. Moreover, on appropriate storage, no activity loss is seen after 60 days. The ability of the BaCel 5A containing RIMER to perform hydrolysis of derivatised cellulose was also investigated using carboxymethyl cellulose (CMC) as substrate. Separation and detection were carried out using size exclusion chromatography (SEC) with refractive index detection (RI). The results show that the RIMERs are robust and can be employed for on-line hydrolysis of both cellodextrins and derivatised cellulose of high molecular weight. (c) 2005 Elsevier B.V. All rights reserved.
- ISSN: 0003-2670