Xyloglucan mobilisation and purification of a (XLLG/XLXG) specific β-galactosidase from cotyledons of Copaifera langsdorffii

Abstract

The storage xyloglucan of germinating seeds of Copaifera langsdorffii is degraded by the action of β-galactosidase, endo-β-glucanase, α-xylosidase and β-glucosidase, producing free galactose, glucose and xylose. One of the β-galactosidases from cotyledons of germinating seeds of C. langsdorffii was purified by ion exchange and gel chromatography (Biogel P-60), leading to a single polypeptide (molecular mass 40 kDa). The enzyme has optimum activity at pH 3.2 (stable from pH 2.3 to 6.0) and is active on p-NP-β-gal (K_m 3.5 mM) and lactose but not on o-NP-β-gal or p-NP-β-gal. Small amounts of galactose were released from xyloglucan of seeds of C. langsdorffii, Tamarindus indica and less from Hymenaea courbaril. No galactose was released after incubation with β-1,4-linked galactan from Lupinus angustifolius cotyledons. Much higher activity was observed on oligosaccharides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichoderma viride cellulase. The purified β-galactosidase attacked XLLG and XLXG specifically, producing a mixture of XXXG and XXLG (unsubstituted glucose is assigned G; glucose branched with xylose is assigned X and if galactose is branching xylose, the trisaccharide is assigned L). Considering the recent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents β-glucosidase from acting on GLXG or GLLG but not on GXLG or GXXG, the β-galactosidase isolated in this work seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance (L) for further hydrolysis of the oligosaccharides and therefore essential for completion of xyloglucan mobilisation.

Introduction

Xyloglucans are cell wall storage polysaccharides predominantly accumulated in cotyledonary tissues of several species such as tamarind (Tamarindus indica) [21], Tropaeolum majus [15], Hymenaea courbaril [3] and Copaifera langsdorffii [6].

Seed xyloglucans have a main β(1→4)-glucan backbone (G) that may be branched with α(1→6)-linked D-xylopyranosyl (X) or β-D-galactopyranosyl(1→2)D-xylopyranosyl (L) [15]. Except for the absence of terminal fucosyl units α(1→2)-linked to the β-D-galactosyl groups, there is a remarkable similarity between seed reserve xyloglucan and structural xyloglucan from primary walls of dicotyledoneous vegetative tissues [19]. The basic polymer molecule is composed of heptasaccharide repeating units – Glc₄:Xyl₃ – with variation in the substitution with galactose residues [32], and also fucose residues on primary walls [19]. Recently, a new repeating unit was found in seeds of Hymenaea courbaril, consisting of Glc₅:Xyl₄ (XXXXG) [5].

Xyloglucans from seeds of T. majus, T. indica and C. langsdorfii are composed of the same four basic structural units XXXG, XLXG, XXLG and XLLG [6], [32]. The ratios between these units give rise to highly specific structures in different species and even between populations of the same species growing in different environments [6]. The polysaccharides of C. langsdorffii seeds from plants grown in the forest contain slightly higher amounts of galactose than the seeds from the cerrado (a savannah-like vegetation). Both, however, present higher galactose branching than the xyloglucan of tamarind. This indicates significant differences in the distribution of residues in the lateral chains and a higher uniformity in the distribution of the repeating units [6].

In cotyledons of T. majus, the rate of xyloglucan mobilisation coincided with the increase and later the decrease in the levels of four hydrolytic enzymes: endo-(1→4)-β-D-glucanase, β-D-galactosidase, α-D-xylosidase and β-D-glucosidase [15]. The endo-(1→4)-β-D-glucanase was purified to homogeneity and showed specificity toward xyloglucans [16]. One β-galactosidase has also been purified to homogeneity from cotyledons of nasturtium. Unlike other β-galactosidases, this enzyme is capable of removing side-chain galactose residues from xyloglucan without any prior depolymerisation of the cellulosic backbone [16]. An α-xylosidase (or exo-oligoxyloglucan-α-xylohydrolase) recognises as substrate exclusively xyloglucan oligosaccharides, taking one xylosyl residue at a time from the non-reducing end of the molecule [17]. Recently, Crombie et al. [10] purified the β-glucosidase from the germinating cotyledons of nasturtium and found that this enzyme is not capable of attacking glucosyl residues at the non-reducing end of the oligosaccharides GLXG and GLLG, but it does hydrolyse GXLG of GXXG. The authors suggest that the galactose present towards the non-reducing end of xyloglucan oligosaccharides is a major constraint to their complete hydrolysis.

Buckeridge et al. [6] inferred the presence of these enzymes in cotyledons of C. langsdorffii through the detection of glucose, galactose and xylose liberated by crude enzymic extracts containing endogenous water soluble xyloglucan.

The similarity of the xyloglucan structure and metabolism in seeds of this native tropical species [6] with those of other studied species, e.g. T. indica [26] and T. majus [15], motivated the present study where, besides the detection of enzyme activities following xyloglucan mobilisation, the purification of one of the β-galactosidases was performed. Its mode of action on xyloglucan and oligomers was studied and its role in storage xyloglucan mobilisation is discussed.