pThe cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (iPopulus tremula/i×itremuloides/i) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast iPichia pastoris/i. Sequence analysis indicated a high degree of similarity with other proteins in the iXTH/i (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in iP. pastoris/i is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the α-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asnsup93/sup to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asnsup93/sup→Ser) mutant./p
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