Xylanase from Trichoderma pseudokoningii: Purification, Characterization, and Effects on Isolated Plant Cell Walls

BAKER, C. J., C. H. WHALEN, and D. F. BATEMAN. 1977. Xylanase from Trichoderma pseudokoningii: purification, characterization, and effects on isolated plant cell walls. Phytopathology 67:1250-1258. Trichoderma pseudokoningii produced an extracellular xylose from /3-1,4 xylan. It did not hydrolyze arabinan endo-31,4 xylanase when grown in shake culture at 25 C on a (araban), carboxymethylcellulose, /3-1,4 galactan, mannan, mineral salts medium containing 0.1% /3-1,4 xylan as the sole glucomannan, sodium polypectate, or polygalacturonic acid. carbon source. Enzyme activity was determined by It had a pH optimum of 5.0, a pI of about 9.6, a molecular measuring the release of reducing groups from xylan or 0weight of 15,000 to 22,000, and was stable in 50 mM sodium hydroxyethyl xylan. Dialyzed culture filtrates from 4-dayacetate buffer (pH 5.0) at -20 C for up to 9 mo. The purified old cultures were subjected to ion exchange chromatography endo-P3-1,4 xylanase readily solubilized carbohydrate on CM-Sephadex (C-25) in 20 mM sodium acetate buffer containing xylose and arabinose from isolated cell walls of 5(pH 5.0); xylanase was eluted with a linear salt gradient (0day-old corn seedlings, and solubilized, to a lesser extent, 150 mM NaCl in buffer). Fractions containing the enzyme carbohydrate containing primarily xylose from isolated cell were dialyzed, lyophilized, dissolved in water, and subjected walls from hypocotyls of 7-day-old bean seedlings. This to gel filtration on Bio-Gel P-10 equilibrated with 50 mM enzyme should be valuable in evaluating the role of xylanase sodium acetate buffer (pH 5.0) containing 100 mM NaCl. in cell wall hydrolysis by pathogens and in helping to further This two-step procedure yielded a 28-fold purification of the elucidate plant cell wall structure. xylanase. The purified enzyme released only oligomers of Additional key words: cell wall degradation, xylan. The D-xylans are polysaccharides found in the pseudokoningii, a nonpathogen, would be a good source hemicellulosic fraction of higher plant cell walls. These for this enzyme. Here we report the purification and polymers consist of a/3-1,4-linked D-xylopyranose chain characterization of an endo-/3-1,4 xylanase (EC 3.2.1.8) which commonly has side branches of a-l,3-linked Lproduced by T.pseudokoningii and present evidence that arabinofuranose and a-1,2-linked D-glucuronopyranose cell wall sugars are released from isolated corn (Zea mays (or its 4-0-methyl ether). The number and kinds of branch L. ) and bean (Phaseolus vulgaris L.) cell walls by this residues are characteristic for different plant groups. xylanase. Preliminary reports of these studies have been Some xylans are acetylated at carbons 2 and 3 of the made (2, 4). xylopyranose residues (35). The D-xylans are found in almost all land plants (31) MATERIALS AND METHODS and they are the principal components of the primary cell walls of monocots (7). Since a number of microorganisms Commercial xylan (larchwood) was obtained from pathogenic to such plants are known to produce ICN Pharmaceuticals, Inc. Cleveland, OH 44128. xylanases (5, 6, 10, 25, 30, 33), it is of interest to know Cytochrome C from horse heart, horseradish peroxidase, whether or not the degradation of these polysaccharides ribonuclease A, and a-chymotrypsinogen A were plays a role in pathogenesis. The degradation of the obtained from Sigma Chemical Co., St. Louis, MO xylopyranosyl chain by microorganisms can be 63178. Ampholines for isoelectric focusing were accomplished through random hydrolytic cleavage of the purchased from LKB Instruments, Inc., Rockville, MD /3-1,4 glycosidic bonds by xylanases (8, 9, 11, 12, 17, 34). 20852. Carboxymethylcellulose (Type 7 MP) was To study the effects of these enzymes on plant cell walls obtained from Hercules Inc., Wilmington, DE 19899. and to evaluate their possible role in pathogenesis, pure Polygalacturonic acid was purchased from the Sunkist enzyme preparations are needed. Such preparations also Growers, Inc., Ontario, CA 91764. Mannan was would serve as powerful tools in the further elucidation of purchased from K & K Laboratories, Plainview, NY the plant cell wall structure. A survey of several 11803, and araban was obtained from Koch-Light microorganisms that produce xylanases that could be Laboratories, Ltd., Colnbrook, U.K. Beta-l,4 galactan purified readily indicated that Trichoderma was purified from Lupinus albus L. by the procedure of Jones and Tanaka (13). Glucomannan was prepared from Copyright © 1977 The American Phytopathological Society, 3340 the commercial xylan as described below. Xylan was Pilot Knob Road, St. Paul, MN 55121. All rights reserved, further purified from the commercial larchwood xylan