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

The storage xyloglucan of germinating seeds of Copaifera langsdorffii is de graded by the action of beta-galactosidase, endo-beta-glucanase, alpha-xylo sidase and beta-glucosidase, producing free galactose, glucose and xylose. One of the beta-galactosidases from cotyledons of germinating seeds of C. l angsdorffii was purified by ion exchange and gel chromatography (Biogel P-6 0), 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-beta-gal (K-m 3.5 mM) and lactose but not on o-NP-beta-gal or p-NP beta- gal. Small amounts of galactose were released from xyloglucan of seeds of C . langsdorffii, Tamarindus indica and less from Hymenaea courbaril. No gala ctose was released after incubation with beta-1,4-linked galactan from Lupi nus angustifolius cotyledons. Much higher activity was observed on oligosac charides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichode rma viride cellulase. The purified beta-galactosidase attacked XLLG and XLX G 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 rec ent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents beta-glucosidase from acting on GLXG or GLLG but not on GXLG or GXXG, the beta-galactosidase isolated in this work seem s to perform a key role in xyloglucan degradation since it is responsible f or the retrieval of a major sterical hindrance (L) for further hydrolysis o f the oligosaccharides and therefore essential for completion of xyloglucan mobilisation. (C) 1999 Editions scientifiques et medicales Elsevier SAS.