Structural Elucidation of Enzymatically Synthesized ... - ACS Publications

Apr 24, 2016 - Karnegijeva 4, 11000 Belgrade, Serbia. §. INRA, UR1268 Biopolymers Interactions Assemblies, F-44316 Nantes, France. #. Faculty of ...
2 downloads 0 Views 1MB Size
Article pubs.acs.org/JAFC

Structural Elucidation of Enzymatically Synthesized Galactooligosaccharides Using Ion-Mobility Spectrometry−Tandem Mass Spectrometry Milica Carević,† Dejan Bezbradica,*,† Katarina Banjanac,† Ana Milivojević,† Mathieu Fanuel,§ Hélène Rogniaux,§ David Ropartz,§ and Dušan Veličković# †

Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia § INRA, UR1268 Biopolymers Interactions Assemblies, F-44316 Nantes, France # Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, Serbia S Supporting Information *

ABSTRACT: Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with β-galactosidases. Enzymatic transgalactosylation results in a mixture of compounds of various degrees of polymerization and types of linkages. Because structure plays an important role in terms of prebiotic activity, it is of crucial importance to provide an insight into the mechanism of transgalactosylation reaction and occurrence of different types of β-linkages during GOS synthesis. Our study proved that a novel one-step method, based on ion-mobility spectrometry− tandem mass spectrometry (IMS-MS/MS), enables complete elucidation of GOS structure. It has been shown that βgalactosidase from Aspergillus oryzae has the highest affinity toward formation of β-(1→3) or β-(1→6) linkages. Additionally, it was observed that the occurrence of different linkages varies during the reaction course, indicating that tailoring favorable GOS structures with improved prebiotic activity can be achieved by adequate control of enzymatic synthesis. KEYWORDS: A. oryzae, galacto-oligosaccharides, structure, IMS-MS/MS, mass spectrometry



synthesis should be focused on fine-tuning of physiological properties. In vitro studies showed that the molecular weight of GOS significantly influences the growth of probiotic bacteria, whereas in a different study it was proved that β-(1→3)- or β(l→6)-linked GOS are preferentially metabolized by gut probiotics.5,19 Hence, it seems reasonable that targeted control of enzymatic processes toward obtaining predominantly GOS of desired length and with preferential type of β-linkage will give further impetus in this field of application. Such a goal is very challenging from a bioanalytical point of view, because adequate reaction optimization and control require the development of methods that quickly and accurately provide information about the concentration of each synthesized GOS isomer. In our study, a novel method, based on an ion-mobility spectrometry−tandem mass spectrometry (IMS-MS/MS) approach, was used for one-step complete analysis of GOS mixtures obtained in lactose transgalactosylation using βgalactosidase from A. oryzae. Due to its significant transgalactosylation activity, this particular enzyme has been widely used in GOS synthesis;20−23 hence, attempts were made to determine the structure of all obtained oligosaccharides. In a study performed by Urrutia et al.24 high-performance anion

INTRODUCTION Prebiotics, being short-chain carbohydrates nondigestible by digestive enzymes in humans with consequential property of promoting growth of beneficial bacteria, are compounds that attract enormous interest in the dairy industry.1,2 Prebiotics give added value to functional dairy products by stimulation of preferential growth of probiotic lactobacilli and bifidobacteria in the human gut, improvement of product taste and texture, and stability of emulsions.3−5 Galacto-oligosaccharides (GOS) are a group of β-linked carbohydrate compounds containing galactose and glucose, produced by enzymatic transgalactosylation of lactose,5,6 hence by far the most compatible prebiotics for application in dairy products. Industrial production of GOS started around 30 years ago in Japan,7 and it expanded rapidly in Japan and Europe, with predominant application in infant nutrition.5,7,8 Industrial processes are performed using βgalactosidases (EC 3.2.1.23), enzymes with the primary biological purpose of catalyzing the hydrolysis of β-galactoside bonds, but with the additional property of catalyzing the synthesis of oligosaccharides in conditions that favor their transgalactosylation activity.6,9,10 β-Galactosidases from different microorganisms, such as Aspergillus oryzae,11,12 Bacillus circulans,13,14 Kluyveromyces lactis,9,15 and Bifidobacterium bifidum,16,17 are applied in these processes, and they promote the synthesis of GOS with various degrees of polymerization (DP) and contribution of different linkages, namely, β-(1→3), β-(1→4), and β-(1→6).18 State of the art in current GOS production implies that further optimization of enzymatic GOS © 2016 American Chemical Society

Received: Revised: Accepted: Published: 3609

March 20, 2016 April 20, 2016 April 24, 2016 April 24, 2016 DOI: 10.1021/acs.jafc.6b01293 J. Agric. Food Chem. 2016, 64, 3609−3615

Article

Journal of Agricultural and Food Chemistry

Figure 1. Time course of overall GOS synthesis (◆) and synthesis of oligosaccharides with different polymerization degrees: GOS DP3 (△) and GOS DP4 (□). galactosidase (0.2 IU/mL) dissolved in 0.1 M sodium acetate buffer (pH 4.5) to reach a total volume of 20 mL. Reaction was monitored for 150 min, and samples were taken throughout the reaction time. The reaction was stopped by heating samples at 100 °C for 10 min to inactivate the enzyme. At the same time, control samples (without enzyme) were prepared and the product was not detected in them. Afterward, samples were 10-fold diluted with HPLC grade water, filtered through a 0.2 μm syringe filter, and then analyzed by HPLC. All experiments were carried out in duplicate, and average values of obtained product concentrations are presented. All standard deviations were