Journal of Molecular Catalysis B: Enzymatic 28 (2004) 45–53 Operation and performance of analytical packed-bed reactors with an immobilised alcohol oxidase A.M. Azevedo a , J.M.S. Cabral a , T.D. Gibson b , L.P. Fonseca a,∗ a Centro de Engenharia Biológica e Qu´ ımica, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal b T&D Technology, 416 Aberford Road, Stanely, Wakefield, West Yorkshire WF3 4AA, UK Received 14 November 2003; received in revised form 23 December 2003; accepted 13 January 2004 Abstract Hansenula polymorpha alcohol oxidase (AOX) was immobilised on propylamino-derivatised controlled pore glass (CPG) by covalent attachment using glutaraldehyde (GA) as cross-linker. Different pore and particle sizes were used as well as different experimental conditions (GA concentration, buffer type, pH, time of activation) to optimise the enzymatic productivity and operational stability of the immobilised enzyme. The best results were obtained by activating CPG with 6.5% GA in phosphate buffer, pH 7, for 1 h. The highest activity (0.45 U/mg) and productivity (6.2 mol/m 3 min) were obtained with a CPG support with 120–200 mesh and 550 Å pore size. Mini packed-bed bioreactors (9.4–69 mm 3 ) with the immobilised AOX, were used to monitor ethanol. The performance of the bioreactors was simulated using a plug flow model. External mass transfer limitations were observed for residence times higher than 2 s. The bioreactors operated continuously at 32 ◦ C for more than 14 h without significant loss of performance (less than 5%). Ethanol in real samples such as beer, brandy and fermentation media was also successfully monitored. Bi-enzymatic bioreactors containing AOX and HRP were further constructed and displayed a similar performance. © 2004 Elsevier B.V. All rights reserved. Keywords: Hansenula polymorpha; Alcohol oxidase; Immobilisation; Glutaraldehyde; Controlled pore glass; Operational stability; Immobilised enzyme reactors; Mass transfer limitations; Horseradish peroxidase 1. Introduction Immobilised enzyme reactors have been widely used in analytical chemistry, offering many advantages over homo- geneous enzyme systems, especially when incorporated into adequately designed flow systems that minimise reagent consumption and allow the handling of small sample vol- umes [1]. Enzymes have been immobilised onto solid supports, ei- ther by physical adsorption, covalent bonding, cross-linking and entrapment [2]. The immobilisation of enzymes en- ables their continuous use in analytical assays, reactors, sen- sors and industrial processes with high retention of activity. Of the methods commonly used, covalent binding onto in- ert supports favours a long-time stability and the re-use of the immobilised enzyme, although the activity may become drastically reduced. Controlled pore glass (CPG) is one of the most popular solid supports for covalent immobilisa- ∗ Corresponding author. Tel.: +351218419065; fax: +351218419062. E-mail address: lfonseca@alfa.ist.utl.pt (L.P. Fonseca). tion. CPG is a macroporous high-silica glass, obtained from alkali-borosilicate glass, a material which has excellent me- chanical properties and can be prepared with a wide range of porosities and pore sizes [3]. The obtention of high activities and stabilities during op- erational conditions usually requires an optimisation of the enzyme immobilisation protocol. On the other hand, the par- ticle and pore size can have a major impact on the activity and stability of the immobilised enzyme. The larger the par- ticle, the greater the effect of diffusion control. Hence, the smallest particle size is usually the best choice, although in this case pressure drop issues become important if the use of a packed bed is envisaged. Regarding the support pores, the smaller their size, the higher the surface area available for enzyme loading. Nevertheless, extremely small pores may exclude the target enzyme [4]. Alcohol oxidase (AOX; E.C. 1.1.3.13; alcohol:O 2 oxi- doreductase) is an oligomeric flavoprotein with eight iden- tical sub-units arranged in a quasi-cubic orientation, each containing a non-covalently bound flavin adenine dinu- cleotide molecule (FAD) as a cofactor [5]. AOX catalyses the oxidation of low molecular weight alcohols by molecular 1381-1177/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2004.01.001