Purification and characterization of three (14)-beta-d-xylan endohydrolases from germinated barley

Summary of procedure.7 used lo purifi, (I +4)-1$,rylan endo- hydroluses from extracts of germinated barley Polybuffer 74/HC1, pH 4 (Pharmacia LKB) (Fig. 4). During the chromatofocusing step, rccovery of xylan hydrolase ac- tivity was less than 5% (Table 1) and most of the protein applied to the column rcmained bound under the conditions uscd (Table 1, Fig. 4). To remove Polybuffer and traces of contaminating proteins, pooled fractions were concentrated by ultrafiltration and simultaneously re-equilibrated in 50 mM sodium acetate, pH 5.0. and applied to a 76cm x 1 cm column of Ultrogel AcA44 (IBF Biotechnics, France) equiIi- brated at 4°C in the same buffer. Fractions containing activity were examined by SDS/PAGE, pooled, concentrated by ultrafiltration and stored at ~ 80°C. Significant losses in ac- tivity were also observed during this gel filtration chro- matography step (Table 1). Carhox2,metlzyl-cellulo~se chromatography. During ultra- filtration, fractions XH2/XH3 from the Procion blue dye chromatography (Fig. 3) were equilibrated in 20 mM sodium acetate, pH 5.0 and applied to a 32.5 cm