Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex

We describe a novel and general, mechanism-based, method for purification o f xyloglucan endotransglycosylases (XETs) from crude plant extracts. Putati ve isoforms, obtained by step-wise precipitation with (NH4)(2)SO4, were inc ubated with tamarind xyloglucan (approximate to 1 MDa) to form stable xylog lucan-XET complexes with apparent molecular masses > 500 kDa on gel-permeat ion chromatography (GPC). Subsequent addition of xyloglucan-derived oligosa ccharides (a mixture of XET acceptor substrates) caused a shift in the GPC elution volume of the activity back to that expected of a approximate to 32 kDa protein, presumably by completing the transglycosylation reaction and so freeing the enzyme from the xyloglucan (donor substrate). This simple tw o-step method enabled the isolation of each XET activity attempted [various (NH4)(2)SO4 cuts from extracts of cauliflower florets and mung bean seedli ngs], in pure form as judged by SDS/PAGE.