Functional interactions among three xylanases from Trichoderma harzianum Ken K. Y. Wong, Larry U. L. Tan, and John N. Saddler Biotechnology and Chemistry Department, Forintek Canada Corp., 800 Montreal Road, Ottawa, Canada K1G 3Z5 (Received 29 January 1986; revised 8 May 1986) The functional importance of xylanase multiplicity in Trichoderma harzianum has been examined by studying the hydrolysis of different forms of xylan. The hydrolyses achieved by xylanase combina- tions were compared with that achieved by the xylanases individually. Complementation among three purified xylanases was observed in the substrate limiting hydrolysis of isolated aspen xylans although it was not observed with oat arabinoxylan. The three enzymes together provided the greatest hydrolysis of aspen xylans, enhancing the hydrolysis of acetylated and deacetylated aspen xylans by 2 7 and 18%, respectively, when compared with hydrolyses obtained with the individual enzymes. The degree of complementation was much higher in the hydrolysis of aspen holocellulose. Combina- tions of two or three xy!anases improved holocellulose hydrolysis by 26-114%. All three enzymes were required to achieve the greatest degree of hydrolysis. Xylanase multiplicity in this organism is required for effective hydrolysis of xylan in complex substrates. It appears that the characterized xylanases are not redundant enzymes since each contributes significantly and uniquely to the xylano- lytic system of the fungus. Keywords: Xylanases; xylanase multiplicity; complementation; Trichoderma harzianum; xylans; holoceUulose Introduction 13-1,4-Xylan, a major polysaccharide in lignocellulose, has a backbone of 3-1,4 linked xylopyranose residues which may be substituted with acetyl residues and residues of arabi- nose and methylglucuronic acid) The complexity of this heteropolymer suggests that several enzymes are required for its complete hydrolysis. Previously, we had reported the purification and characterization of three xylanases (1,4- ~-D-xylan xylanohydrolase, EC 3.2.1.8) from the fungus Trichoderma harzianum E58. 2-4 The amino acid composi- tions, physicochemical properties and substrate specifici- ties of these enzymes suggested that each was a distinct gene product which was produced specifically for the hydro- lysis of xylan. It is not known whether each enzyme contri- butes unique and essential functions to enable a more effec- tive utilization of the substrate by the organism. This ques- tion also addresses concerns in the biotechnology field where effective hydrolysis of biomass is sought. Xylanase multiplicity has been studied most extensively in Aspergillus niger. Takenishi and Tsujisaka 5 elucidated the functional importance of two xylanases purified from a strain of A. niger. They found that these two xylanases complemented each other to increase the extent of hydro- lysis in an arabinoxylan. One enzyme released the arabinose substituent from arabinosylxylotriose and the other hydro- lysed xylotriose. Similar properties have been found in other pairs of xylanases purified from A. niger 6 and Cera- tocystis paradoxa. 7's However, the importance of higher degrees of xylanase multiplicity has not been well evaluated. Takenishi and Ysujisaka s purified a third xylanase from A. niger; however, their hydrolysis experiments suggested that it was a redundant enzyme. Other workers found that a mixture of all three xylanases purified from Talaromyces byssochlamydoides increased the extent of hydrolysis of an arabinoxylan. 9 The specific activity of a mixture of three xylanases from Streptomyces exfoliatus was greater than that calculated from their individual specific activi- ties. 1° In the last two cases, hydrolyses achieved by combi- nations of two enzymes were not reported. Different forms of xylan must be considered to assess the functional importance of high degrees of xylanase mul- tiplicity. In this work, we have studied the functional interactions among three xylanases from T. harzianum by measuring the hydrolysis of various types of isolated xylans and xylan in holocellulose. Materials and methods Subs tra tes Aspen holocellulose was prepared from extractive-free aspen sawdust (~< 20 mesh) using a chlorite treatment, u The residue obtained after 2 h of treatment was ground to pass through a 1 mm mesh and subsequently treated for a further 2 h. The yield of holocellulose from extractive- free aspen sawdust was 84%. 0141 --0229/86/100617--06 $03.00 © 1986 Butterworth & Co. (Publishers) Ltd Enzyme Microb. Technol., 1986, vol. 8, October 617