Substrate specificity of novel GH16 endo-β-(1→3)-galactanases acting on linear and branched β-(1→3)-galactooligosaccharides

Highlights

• Three novel endo-beta-1,3 galactanases and their activities are reported.

• Substrate specificity is studied using well-defined, branched and linear oligosaccharides.

• The relative activity and the products formed are compared between the enzymes.

Abstract

Arabinogalactan proteins are proteoglycans located in the plant cell wall. Most arabinogalactan proteins are composed of carbohydrate moieties of β-(1→3)-galactan main chains with β-(1→6)-galactan side chains terminated by other glycans. In this study, three novel endo-β-(1→3)-galactanases were identified and the substrate specificity was further studied using well-defined galactan oligomers. Linear and branched β-(1→3)-linked galactans, which resemble the carbohydrate core of the arabinogalactan protein, were used for the characterization of endo-β-(1→3)-galactanases. The identified enzymes required at least three consecutive galactose residues for activity. Non-substituted regions were preferred, but substituents in the -2 and +2 and in some cases also -1 and +1 subsites were tolerated to some extent, depending on the branching pattern, however at a significantly lower rate/frequency.

Introduction

Arabinogalactan proteins (AGPs) belong to the family of hydroxyproline-rich glycoproteins (HRGPs) and they are characterized as highly glycosylated proteoglycans (Pereira et al., 2015). They are considered as one of the most complex macromolecule families found in plants. Their complexity is revealed from the remarkable diversity of the glycans attached to the protein backbone, compromising 90–95% of the total mass (Ellis et al., 2010). Determining the precise structure of the carbohydrate moiety of the AGPs is of major importance to better understand their biosynthesis and their biological and possible industrial applications (Ling et al., 2012). AGPs are known to be involved in various processes in plant growth and development such as cell division (Serpe and Nothnagel, 1994), cell development (Van Hengel and Roberts, 2002), signaling and cell death (Gao and Showalter, 1999).

AGPs consist of a core protein rich in hydroxyproline and large arabinogalactan (AG) domains that commonly comprise a backbone of β-(1→3)-linked d-galactose with branch points of β-(1→6)-linked d-galactose of one, two or three residues in length usually terminating in arabinofuranose (Araf), rhamnopyranose (Rhap) and galactopyranose (Galp) residues (Ellis et al., 2010). Some AGs are additionally decorated by short Araf oligosaccharide chains and others are rich in uronic acid residues (GalpA, GlcpA) (Showalter, 2001).

AGPs can be used as food and pharmaceutical products due to their general adhesive, emulsifying and water-retaining properties (Showalter, 2001). They are important components of various plant gums or exudates and bestow remarkable properties on these plant products. For instance, gum arabic is a dried exudate from Acacia Senegal and represents one of the most commercially significant gums. It is widely used in the pharmaceutical and food industries due to its low viscosity and toxicity (Showalter, 2001). Gum arabic is a polydisperse molecule, which has morphological similarities with the AGP complex and AG fractions (Nie et al., 2013). By investigating its structure with 2D NMR spectroscopy, it was proposed that gum arabic is a branched complex polysaccharide, which constitutes of a backbone of β-1,3-galactan with extensive branching at the O-2, O-4 or O-6 position. The branches consist of β-galactosyl, β-glucuronosyl, α-arabinosyl, and α-rhamnosyl residues (Mahendran et al., 2008).

Enzymes hydrolyzing β-1,3-galactans include exo-β-(1→3)-galactanases and endo-β-(1→3)-galactanases. Exo-β-(1→3)-galactanases from glycosyl hydrolase family 43 (GH43) hydrolyze the β-(1→3)-galactan backbone of e.g. partially debranched gum arabic or larchwood arabinogalactan in an exo fashion requiring at least two consecutive galactose residues for activity. Remarkably, they are able to bypass branch points liberating also intact β-(1→6)-linked galactooligosaccharide side chains (Ling et al., 2012; Sakamoto and Ishimaru, 2013; Tsumuraya et al., 1990). Endo-β-(1→3)-galactanase activity was observed already around 1970 (Hashimoto, 1971; Hashimoto et al., 1969), but has been much less studied and was first rigorously characterized by Kotake and co-workers in 2011 (Kotake et al., 2011). The authors identified the first endo-β-(1→3)-galactanase (FvEn3GAL) from the winter mushroom Flammulina velutipes which was subsequently cloned, purified and characterized. Two homologous endo-β-(1→3)-galactanases (45–46% sequence homology to FvEn3GAL) from Aspergillus flavusi (Af3G) and from Neurospora crassa (NcEn3GAL) were recently expressed in Pichia pastoris, purified and characterized (Yoshimi et al., 2017). All three enzymes specifically hydrolyse β-(1→3)-galactan and β-(1→3)-galactooligosaccharides with degree of polymerization of at least three (Kotake et al., 2011; Yoshimi et al., 2017). However, the enzymes only have trace activity towards native gum arabic, probably because branching at the 6-position prevents them from accessing the β-(1→3)-galactan main chains. These enzymes are classified as EC 3.2.1.181 and belong to GH16 (Sakamoto and Ishimaru, 2013), a family that also includes endo-β-(1→3(4))-glucanases. Apart from FvEn3GAL, NcEn3GAL and Af3G, no other endo-β-(1→3)-galactanases have been described in the literature. Endo-β-(1→3)-galactanases could be relevant industrially due to their potential to process AGP, gum arabic or larchwood arabinogalactan, possibly in combination with auxiliary, debranching enzymes such as endo-β-(1→6)-galactanases, α-l-arabinofuranosidases and α-l-rhamnosidases (Sakamoto and Ishimaru, 2013; Yoshimi et al., 2017).

In the present study, three novel fungal endo-β-(1→3)-galactanases from GH family 16 were identified and their modes of action were investigated by studying their activity on well-defined linear and branched AGP oligosaccharides produced by chemical synthesis (1–4, Fig. 1).