Please cite this article in press as: C. Vera, et al., Synthesis of propyl--d-galactoside with free and immobilized -galactosidase from Aspergillus oryzae, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.11.024 ARTICLE IN PRESS G Model PRBI-10869; No. of Pages 10 Process Biochemistry xxx (2016) xxx–xxx Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Synthesis of propyl--d-galactoside with free and immobilized -galactosidase from Aspergillus oryzae Carlos Vera ∗ , Cecilia Guerrero, Lorena Wilson, Andrés Illanes School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2085, Valparaíso, Chile a r t i c l e i n f o Article history: Received 23 August 2016 Received in revised form 4 November 2016 Accepted 30 November 2016 Available online xxx Chemical compounds studied in this article: 2-Nitrophenyl--d-galactopyranoside (PubChem CID: 96647) -Lactose (PubChem CID: 84571) 1-Propanol (PubChem CID: 1031) Keywords: Propyl-galactoside -Galactosidase Enzyme immobilization Sugar-based surfactant Alkyl-galactosides a b s t r a c t Synthesis of propyl--galactoside catalyzed by Aspergillus oryzae -galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Opti- mal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl--galactoside was evaluated at the optimal condition previously determined, with immobilized -galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl--galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl--galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allow- ing catalyst reuse, and avoiding browning reactions. Propyl--galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry. © 2016 Published by Elsevier Ltd. 1. Introduction -Galactosidases catalyze the hydrolysis of -glycosidic bonds in terminal and non-reducing -galactosides. For decades, its main use has been in the hydrolysis of lactose in milk and dairy products [1]. However, -galactosidases can also catalyze trans- galactosylation reactions by transferring a galactose residue to a hydroxyl-containing nucleophile [2]. This latter potential began to be exploited commercially more than a decade ago for the pro- duction by transglycosylation of prebiotic galacto-oligosaccharides and other non-digestible oligosaccharides derived from lactose [2–4]. Alkyl glycosides (AGs) are other compounds that can be produced by transglycosylation [5,6]. AGs are non-ionic, biodegrad- able, hypoallergenic and chemically stable surface-active agents. They are stable in acid and alkaline media and are non-reactive in the presence of oxygen being ideal ingredients in personal care, cosmetics, foods and pharmaceutical products [5–7]. AGs are con- formed by a carbohydrate hydrophilic head and a hydrophobic hydrocarbon tail usually derived from a primary fatty alcohol [6]. ∗ Corresponding author. E-mail address: carlos.vera@pucv.cl (C. Vera). Interfacial behavior of AGs can be tuned in an ample range accord- ing to the nature of the carbohydrate head, the length of the alkyl chain and the type of glycosidic bond [8]. AGs are chemically pro- duced by Fisher glycosylation, which is the technology of current use being a straightforward and economic process in which the carbohydrate head is reacted with a fatty alcohol in the presence of an acid catalyst at high temperature. The reaction is poorly regio- and stereoselective so that a complex mixture of AGs is formed due to the presence of equally reactive hydroxyl groups. The reac- tion product is formed by  and -alkyl glycoside isomers with varying degrees of polymerization [9]. Anomerically pure -alkyl glycosides can be produced chemically by the method of; Koenigs- Knorr however, synthesis is cumbersome and requires heavy metal salts as halophilic promoter, which has to be exhaustively removed from the product and adequately disposed. Several research groups have assessed the potential of -glycosidases as catalysts for the synthesis of anomerically pure -alkyl glycosides [5,6]. Advan- tages of enzymatic biocatalysis are high volumetric productivity, mild reaction conditions, absence of toxic compounds, less amount and highly biodegradable waste [6,9]. Also, enzymatic processes can be quite advantageous from an environmental perspective in terms of E-factor and atom economy [10]. The former term cor- responds to the mass of waste generated per unit mass of the http://dx.doi.org/10.1016/j.procbio.2016.11.024 1359-5113/© 2016 Published by Elsevier Ltd.