Please cite this article in press as: F. Carvalho, P. Fernandes, Packed bed enzyme microreactor: Application in sucrose hydrolysis as proof-of-concept, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.04.023 ARTICLE IN PRESS G Model BEJ-6198; No. of Pages 8 Biochemical Engineering Journal xxx (2015) xxx–xxx Contents lists available at ScienceDirect Biochemical Engineering Journal jo ur nal home page: www.elsevier.com/locate/bej Packed bed enzyme microreactor: Application in sucrose hydrolysis as proof-of-concept Filipe Carvalho a , Pedro Fernandes a,b,∗ a iBB – Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal b Faculdade de Engenharia, Universidade Lusófona de Humanidades e Tecnologias, Av. Campo Grande, 376, 1749-024 Lisboa, Portugal a r t i c l e i n f o Article history: Received 31 January 2015 Received in revised form 1 April 2015 Accepted 29 April 2015 Available online xxx Keywords: Packed bed bioreactors Immobilized enzymes Enzyme technology Sucrose Continuous operation Silane-coated silica carriers a b s t r a c t A continuous flow enzyme microreactor was designed, assembled and run that allowed for operation with immobilized enzymes in particulate form. As a proof-of-concept, invertase was covalently bound to silane-coated silica carriers and used for the hydrolysis of sucrose into invert sugar syrup. Once glutaralde- hyde solution and enzyme load were optimized, and the kinetic behavior of the immobilized biocatalyst was established, the particles were loaded in the sandwich type microreactor, resulting in a packed bed form. The microreactor was fed with substrate solution within 1.0–8.0% (w/v), at flow rates ranging from 17.5 to 259.0 L/min. Apparent kinetics were evaluated using the Lilly–Hornby model. Accordingly K m(app) values decreased with increasing flow rates, whereas, for higher flow rates, the K m(app) tends to stabilize at values close to that observed for the enzyme in the free form. Full conversion was observed up to 8.0% (w/v) of sucrose. Moreover, the immobilized invertase formulation packed in the microreactor displayed high operational stability, as it retained roughly 100% of its initial activity during 30 days. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Over the last decade, a consistent trend towards the use of microscale processing techniques in biocatalysis has emerged and has been contributing to speed up the development of enzyme based systems [1–3]. The high level of parallelization that is achieved in microfluidic devices allows the high through- put required at the different phases of bioprocess development. Operation in microfluidic environment is characterized by low reagent consumption and energy requirements, concomitantly contributing to reduce the cost of process development and the environmental impact. Moreover, the minute diffusion lengths result in enhanced heat and mass transfer, which coupled to the typical continuous mode of operation under laminar flow, allow for a better control over process conditions and enhanced safety. Finally, production scale may be achieved by numbering-up rather ∗ Corresponding author at: iBB – Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lis- boa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Tel.: +351 218419594; fax: +351 218419062. E-mail addresses: pedro.fernandes@tecnico.ulisboa.pt, Pedroefe@yahoo.com (P. Fernandes). than scaling-up. Altogether, the outcome is a faster transfer of the development stage into production [2–4]. The present work is within the context of the use of microscale platforms on the development of a biocatalytic con- tinuous process anchored in invertase immobilization. Invertase (-fructofuranosidase, EC 3.2.1.26) is mainly used to catalyze the hydrolysis of sucrose in the production of an equimolar mixture of glucose and fructose (invert sugars) that is 20% sweeter and less prone to crystallization than sucrose [5,6]. Invert sugars are widely used in bakery and pastry, where shelf life is improved; in the man- ufacture of artificial honey; and as plasticizing agent in cosmetics [7–9]. Continuous flow enzyme reactions require enzyme immobi- lization, and among the diverse methodologies used for invertase immobilization [10], some have also been used in the design of continuous hydrolytic systems, yet overlooking the potential of microreactors. Thus, Albertini et al. immobilized invertase on glass- ceramic support and proceeded with experiments in a packed bed reactor with alternate-flow [11]. Tomotani and co-workers evaluated the performance of a membrane reactor with invertase adsorbed on anionic polystyrene beads [7]. Invertase, chemically modified with chitosan, was immobilized on pectin coated chitin support by Gómez et al. and packed into a column reactor [12]. Cadena et al. covalently immobilized invertase on polyurethane rigid adhesive foam and subsequently covered the internal sur- face of a metallic column. The resulting structure was used on the http://dx.doi.org/10.1016/j.bej.2015.04.023 1369-703X/© 2015 Elsevier B.V. All rights reserved.