Post-translational processing of beta-d-xylanases and changes in extractability of arabinoxylans during wheat germination.

Endo-1,4-beta-d-xylanase (EC 3.2.1.8, beta-d-xylanase) activity, and arabinoxylan (AX) level and extractability were monitored for the first time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to 24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3-4 DPI). This increase was not accompanied by the discernible presence of homologues of the barley X-I beta-d-xylanase as established by immunodetection. This suggests that other, yet unidentified beta-d-xylanases operate in this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of unprocessed 67 kDa X-I like beta-d-xylanase, which was much more abundant in the presence of GA. The occurrence of higher levels of the unprocessed enzyme was related with higher beta-d-xylanase activities and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa beta-d-xylanase. During the late stage (up to 24 DPI) gradual processing of the 67 kDa beta-d-xylanase occurred and was associated with a drastic increase in beta-d-xylanase activity. Up to 120-fold higher activity was recorded at 24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during the late stage, suggesting that the beta-d-xylanase is processed into more active forms to achieve extensive AX breakdown.

Unprocessed barley aleurone endo-beta-1,4-xylanase X-I is an active enzyme.

Endo-beta-1,4-xylanase X-I is a major hydrolase produced by the aleurone tissue of germinating barley grain. It was previously reported that this cytosolic enzyme is synthesized as an inactive precursor which is proteolytically processed to active forms upon its programmed cell death dependent release. We here demonstrate, however, that the precursor form of X-I is an active enzyme. Purified recombinant precursor X-I was characterised with respect to its molecular weight, iso-electric point and temperature and pH activity and stability. Analysis of the hydrolysis products showed that it is an endo-acting enzyme which has the striking ability to release xylose from both polymeric xylan as well as from small xylo-oligosaccharides. The implications of these findings in relation to the putative role of the N-terminal propeptide as a carbohydrate binding module and the possible consequences for the way X-I fulfils its role in the germination process, are discussed.