Purification of an arabinofuranosidase and two β-xylopyranosidases from germinated wheat

Arabinosidase and P-xylosidase activities were detected in germinated wheat grain, and both increased over seven days of germination, under malting conditions. Arabinosidase was partially purified by anion exchange chromatography, chromatofocusing and gel filtration chromatography. The pH optimum of the partially purified enzyme was 4.2 and the K-M was 1.90 mM p-NP-Ara. Edman degradation, MALDI-TOF mass spectrometry and nano-ESI mass spectrometry were used to identify the two major proteins in the partially purified arabinosidase mixture. The two proteins were a beta-amylase with an amino acid sequence partially homologous to a barley beta-amylase, and three wheat serine protease inhibitors. Further purification, by affinity chromatography and hydrophobic interaction chromatography, removed the identified contaminating proteins. At this point an 80 kDa protein was detected by SDS-PAGE. No identity could be assigned to this protein by MALDI-TOF mass spectrometry by reference to electronic protein databases. Similarly, the beta-xylosidase was partially purified by anion exchange chromatography followed by chromatofocusing and gel filtration chromatography. The first step separated the mixture into two distinct fractions with K-M values of 3.35 and 4.01 mM p-NP-Xyl and pH optima of 4.5. The latter fraction also displayed xylanase activity against RBB-xylan.