Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Short communication Reaction mechanism of β-apiosidase from Aspergillus aculeatus Vladimír Mastihuba ⁎ , Elena Karnišová Potocká, Iveta Uhliariková, Peter Kis, Stanislav Kozmon, Mária Mastihubová Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia ARTICLE INFO Keywords: Apiosidase Reaction mechanism Inverting glycosidase NMR Viscozyme L ABSTRACT Apiosidases are glycosidases relevant for aroma development during fermentation of wines and black tea. Reaction mechanism of apiosidase from Aspergillus aculeatus in commercial glycanase Viscozyme L was studied by 1 H NMR technique. Study of hydrolysis of 4-nitrophenyl β-D-apiofuranoside revealed that this reaction proceeds with inversion of hydroxyl group in the anomeric center, which confirms inverting mechanism of the enzyme and its inability to catalyze transapiosylation in syntheses of apiosides. 1. Introduction D-Apiose is a branched pentose widely spread among vascular plants and aquatic monocots. Although never found in nature as a free sugar, it occurs either as a crosslinking and branching sugar unit of plant cell wall polysaccharides (Loomis & Durst, 1992; O’Neill et al., 1996; Chauvin, Nepogodiev, & Field, 2004; Nepogodiev, Fais, Hughes, & Field, 2011) or as a constituent of plant secondary metabolites pos- sessing various biological activities (Koike, Li, Liu, Hata, & Nikaido, 2005; Kernan et al., 1998; Shiraga et al., 1988; Kaur, Kaur, Kumar, Singh, & Kumar, 2009). D-Apiose is also a frequent constituent of aroma glycosides which are relevant to flavor development in Muscat wines and to a limited extent in tea during the fermentation process (Bayonove et al., 1992; Maicas & Mateo, 2005; Takeo, 1981; Wang, Kurasawa, Yamaguchi, Kubota, & Kobayashi, 2001). D-Apiose in these glycosides is not directly bound to the volatile aglycones but occurs attached to the primary hydroxyl of β-glucosides of flavor active alco- hols to form acuminosides, similarly to xylose and arabinose in aroma- binding primeverosides and vicianosides (Maicas & Mateo, 2005). For example, monoterpene glycosides are the most significant aroma pre- cursors in Alexandria Muscat grapes and wine, binding about 90% of the total monoterpenes (Park, Morrison, Adams, & Noble, 1991). The hydrolysis of aroma glycosides can be enzymatically catalyzed by gly- cosidases either as one-step process in which the aglycone and the free disacharide are released by action of a diglycosidase/endoglycosidase (Günata et al., 1998; Mizutani et al., 2002; Mazzaferro & Breccia, 2011), or in two steps when a specific glycosidase (β-D-apiosidase, β-D- xylosidase, α-L-arabinosidase, α-L-rutinosidase) firstly releases the terminal monosaccharide of the respective diglycoside and the remaining β-glucoside is hydrolyzed by β-glucosidase (Spagna, Barbagallo, Greco, Manenti, & Pifferi, 2002; Günata, Bitteur, Brillouet, Bayonove, & Cordonnier, 1988; Sarry & Günata, 2004). β-D-Apiofuranosidases (β-apiosidases) are glycosidases liberating β- D-apiofuranose residues from the side-chains of rhamnogalacturonan II or from plant glycosides. Their practical use is yet limited to wine- making processes and studies of their occurence and catalytic proper- ties come almost exclusively from the group of Günata (Günata et al., 1988; Sarry & Günata, 2004; Dupin et al., 1992; Guo, Salmon, Baumes, Tapiero, & Günata, 1999; Günata, Dugelay, Valuer, Sapis, & Bayonove, 1997; Günata et al., 1996; Pičmanová & Møller, 2016) for purposes of wider application in wine technology. Revival of interest in apiosidases evolves nowadays due to their potential as auxiliary enzymes in pro- cessing of lignocellulosic biomass. Although there is no pure β-apiosi- dase on the market, our recent study proved that apiosidase side-ac- tivity is surprisingly abundant as secondary activity among industrial enzyme cocktails produced by aspergilli (Kis, Potocká, Mastihuba, & Mastihubová, 2016). Viscozyme L contains high levels of β-apiosidase and this crude enzyme was very recently used in studies of cell wall polysaccharides of aquatic monocots (Avci, Peña, & O’Neill, 2018.). With easily available apiosidase in hands, we tested its potential in preparation of natural β-apiosides and apiose-containing glycosides by the process of transapiosylations from 4-nitrophenyl β-D-apiofurano- side. Since we have observed only hydrolysis of the substrate without formation of any product of transglycosylation, we were particularly interested in studying the reaction mechanism of apiosidase to assess whether the hydrolysis of glycoside bond occurs with eventual reten- tion or inversion of anomeric configuration of the released D-Apiose. Only glycosidases with retaining mechanism are able to catalyze the https://doi.org/10.1016/j.foodchem.2018.09.003 Received 23 May 2018; Received in revised form 29 August 2018; Accepted 1 September 2018 ⁎ Corresponding author. E-mail address: vladimir.mastihuba@savba.sk (V. Mastihuba). Food Chemistry 274 (2019) 543–546 Available online 01 September 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved. T