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Physiology and Biochemistry

Xylanase from Trichoderma pseudokoningii: Purification, Characterization, and Effects on Isolated Plant Cell Walls. C. J. Baker, Graduate Student, Department of Plant Pathology, Cornell University, Ithaca, NY 14853; C. H. Whalen(2), and D. F. Bateman(3). (2)(3)Technician, and Professor and Chairman, respectively, Department of Plant Pathology, Cornell University, Ithaca, NY 14853. Phytopathology 67:1250-1258. Accepted for publication 14 April 1977. Copyright © 1977 The American Phytopathological Society, 3340 Pilot Knob Road, St. Paul, MN 55121. All rights reserved.. DOI: 10.1094/Phyto-67-1250.

Trichoderma pseudokoningii produced an extracellular endo-β-1,4 xylanase when grown in shake culture at 25 C on a mineral salts medium containing 0.1% β-1,4 xylan as the sole carbon source. Enzyme activity was determined by measuring the release of reducing groups from xylan or 0-hydroxyethyl xylan. Dialyzed culture filtrates from 4-day-old cultures were subjected to ion exchange chromatography on CM-Sephadex (C-25) in 20 mM sodium acetate buffer (pH 5.0); xylanase was eluted with a linear salt gradient (0-150 mM NaCl in buffer). Fractions containing the enzyme were dialyzed, lyophilized, dissolved in water, and subjected to gel filtration on Bio-Gel P-10 equilibrated with 50 mM sodium acetate buffer (pH 5.0) containing 100 mM NaCl. This two-step procedure yielded a 28-fold purification of the xylanase. The purified enzyme released only oligomers of xylose from β-1,4 xylan. It did not hydrolyze arabinan (araban), carboxymethylcellulose, β-1,4 galactan, mannan, glucomannan, sodium polypectate, or polygalacturonic acid. It had a pH optimum of 5.0, a pI of about 9.6, a molecular weight of 15,000 to 22,000, and was stable in 50 mM sodium acetate buffer (pH 5.0) at –20 C for up to 9 mo. The purified endo-β-1,4 xylanase readily solubilized carbohydrate containing xylose and arabinose from isolated cell walls of 5-day-old corn seedlings, and solubilized, to a lesser extent, carbohydrate containing primarily xylose from isolated cell walls from hypocotyls of 7-day-old bean seedlings. This enzyme should be valuable in evaluating the role of xylanase in cell wall hydrolysis by pathogens and in helping to further elucidate plant cell wall structure.