Purification of an Arabinofuranosidase and Two β-Xylopyranosidases from Germinated Wheat

Abstract

Arabinosidase and ␤-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-NPAra. 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 ␤-amylase with an amino acid sequence partially homologous to a barley ␤-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 ␤-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 RBBxylan.

Beer and Arabinoxylan

“…There are a number of isoforms of the enzyme and there is variation due to variety ( Slade et al ., 1989 ;Debyser et al ., 1997 ;Autio et al ., 2001 ) and species ( Autio et al ., 2001 ;Simpson et al ., 2003;Grant et al ., 2003 ). It would appear that (1 → 4)-␤ -endoxylanase is the main enzyme that cleaves internal bonds of the xylan backbone.…”

“…Several reports also describe the differences between barley varieties, species and the effect of germination ( Taiz and Honigman, 1976 ;Autio et al ., 2001 ;Grant et al ., 2003 ). Several reports also describe the differences between barley varieties, species and the effect of germination ( Taiz and Honigman, 1976 ;Autio et al ., 2001 ;Grant et al ., 2003 ).…”

Genome-wide association study (GWAS) of seed germination-related traits in rice and identification of candidate genes

“…Other factors such as cGMP, calcium, calmodulin, and protein kinases regulate the aleurone response to hormones during germination (Ritchie et al 2000). In addition to the starch-degrading enzymes, other enzymes that break down cell wall components, including enzymes such as arabinoxylan hydrolases, xylopyranosidase, arabinofuranosidase, β-glucanases, xylanases, endo-mannanase, α-galactosidase and endoxylanase have also been shown to be increased during germination (Guzmán-Ortiz et al 2019;Ha et al 2018;Andriotis et al 2016;González-Calle et al 2015;Gomez et al 2009;Grant et al 2003;Leubner-Metzger, 2003;Caspers et al 2001). Catalase has been suggested to be involved in the promotion of dormant seed germination (Ayşe et al 2012).…”