Biochemical Engineering Journal 82 (2014) 158–165 Contents lists available at ScienceDirect Biochemical Engineering Journal journal h om epage: www.elsevier.com/locate/bej A new mechanism and kinetic model for the enzymatic synthesis of short-chain fructooligosaccharides from sucrose Roberto Vega a,b , M.E. Zuniga-Hansen a,c,∗ a School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2147, Valparaíso, Chile b Department of Biochemistry, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Jr. Puno 1002, Lima, Peru c Centro Regional de Estudios en Alimentos Saludables, Conicyt-Regional, Gore Región de Valparaíso, R06i1004, Blanco 1623, Oficina 1402, Valparaíso, Chile a r t i c l e i n f o Article history: Received 10 April 2013 Received in revised form 13 November 2013 Accepted 16 November 2013 Available online 22 November 2013 Keywords: Enzyme biocatalysis Kinetic parameters Modeling Fructooligosaccharides Multi-response regression Sucrose a b s t r a c t A kinetic model based on a ping-pong mechanism was developed under the steady-state hypothesis to account for the short-chain fructooligosaccharides (sc-FOS) synthesis using the commercial cellulolytic enzyme preparation, Rohapect CM. This new mechanism takes into account the interactions between the enzyme species and potential substrates (sucrose and sc-FOS) as a single complex reaction, allowing a better understanding of the reaction kinetics. The initial reaction rate laws appropriately describe the kinetic profiles of the examined substrates. Whereas sucrose exhibited Michaelis–Menten behavior with substrate inhibition, 1-kestose and nystose followed Michaelis–Menten and sigmoid enzyme kinetics. In addition, the enzyme was competitively inhibited by glucose and exhibited significant hydrolytic activity in the presence of nystose. The overall model was simultaneously fitted to experimental data from three initial sucrose concentra- tions (0.5, 1.5 and 2.1 M) using a multi-response regression with kinetic parameters that have biochemical relevance and are independent of the enzyme concentration. According to the model, sucrose acts almost exclusively as a fructosyl donor substrate. The mathematical development described herein is expected to be suitable for modeling similar enzymatic reaction systems. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Due to the health benefits associated with the consumption of short-chain fructooligosaccharides (sc-FOS), there is increased interest in the use of these compounds in food products [1–3]. As these benefits have been documented for more than two decades, sc-FOS are regarded as prebiotics and granted GRAS status [4]. sc-FOS are fructose oligomers mainly composed of 1-kestose, nystose and 1 F -fructofuranosylnystose, which are obtained from sucrose in the presence of fructosyltransferases (- fructofuranosidase, EC 3.2.1.26 or -d-fructosyltransferase, EC 2.4.1.9). The transfructosylation of sucrose takes place via the cleavage of the -2,1-glycosidic bond and the transfer of the fruc- tosyl moiety onto any acceptor other than water, such as sucrose or a fructooligosaccharide. This synthesis is a complex process in which several reactions occur simultaneously, both in paral- lel and in series, because sc-FOS are also potential substrates of ∗ Corresponding author. Tel.: +56 32 2273650; fax: +56 32 2273803. E-mail addresses: rvegap@unmsm.edu.pe, rovegapa 25@yahoo.es (R. Vega), mzuniga@ucv.cl (M.E. Zuniga-Hansen). fructosyltransferases [5]. Despite this complexity, several kinetic models [6–10] have been developed to predict the reaction progress for the synthesis of sc-FOS from sucrose, and the models are of great interest for defining strategies that allow the optimization and industrial scale-up of these bioprocesses [11–13].sc-FOS are produced from a set of transfructosylation chain reactions that may follow the conventional Michaelis–Menten mechanism [6–9] and the rapid equilibrium random sequential mechanism [10]. Because, these reactions have two reactants, the Michaelis–Menten mechanism is not applicable. Similarly, the second mechanism is also questionable because a covalent fructosyl-enzyme com- plex has been isolated and characterized [14]. In addition, the active site of fructosyltransferases contains a pocket that accom- modates a single sucrose molecule in the substrate-bound structure [15–17]. Furthermore, the rate equations based on the abovemen- tioned mechanisms can only partially describe the progress of the reaction because both make the assumption that the same sub- strate is acting as a donor and acceptor for the fructosyl moiety [6–9]. Alternatively, empirical mechanisms have been proposed to account for the concentrations of reactants and products dur- ing the reaction [18,19]. However, the proportionality constants are a function of operating parameters, including but not 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.11.012