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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.

Additional keywords: cell wall degradation, xylan.