Purification of an Arabinofuranosidase and
Two β-Xylopyranosidases from Germinated Wheat
Melissa M. Grant 1,3, Dennis E. Briggs 1, Colin S. Fitchett 2,
Elaine Stimson 2 and Michael J. Deery 2
1Malting and Brewing Group, Department of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, England,
2 Cereals Innovation Centre, DuPont (UK) Ltd., Station Road, Cambridge, CB1 2UJ, England.
3 Corresponding author. Email: m.m.grant@aston.ac.uk
Current addresses of authors:Melissa M. Grant, LHS, Aston University, Aston Triangle, Birmingham, B4 7ET.Dennis E. Briggs, 66 Sandhills Lane, Barnt Green, Birmingham, B45 8NX, UK.
Colin S. Fitchett, Cambridge Biopolymers Ltd, 13 Sedgwick Street, Cambridge, CB1 3AJ.
Elaine Stimson, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW.
Michael J. Deery, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW.
J. Inst. Brew. 109(1), 8-15, 2003 | VIEW ARTICLE
ABSTRACT
Arabinosidase and β-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 KM 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 β-amylase with an amino acid sequence partially homologous to a barley β-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 β-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 KM 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.
Key words:
Arabinofuranosidase, germinated, wheat, purification, xylopyranosidase.
Publication no. G-2003-0221-133 © 2003 The Institute & Guild of Brewing