Inexpensive, Rapid Procedure for Bulk Purification of Cellulase-Free P-l,4-~-Xylanase of High Specific Activity Larry U. zyxwvutsr L. Tan,* Ernest K. C. Yu, Gerald W. Louis-Seize, and John N. Saddler zyxwvutsr Biotechnolog y and Chemistry Department, Forintek Canada Corp., 800 Montreal Road, Ottawa, Canada Accepted for publication July zyxwvutsr 7 , 1986 A process has been developed for the bulk purification of cellulase-free P-1,4-o-xylanase from the fungus Tri- choderma harzianum E58. The process involved the pri- mary step of ultrafiltering the culture filtrate via a 10,000- molecular-weight cut-off membrane to separate the cel- lulase (retentate) and xylanase (permeate) fractions. The cellulase component was concentrated by 40- to 60-fold, resulting in an enzyme complex that could effectively hydrolyze high concentrations of cellulose and xylan to glucose and xylose. The xylanase was concentrated and solvent exchanged by adsorption to a cationic exchanger, SP-ZetaPrep 250, followed by elution with a pH change in the buffer to give a purified and concentrated xylanase complex dissolved in a low-salt buffer. The resultant xy- lanase system was pure by the criteria of sodium dodecyl sulfate polyacrylamide electrophoresis, had a very high specific activity zyxwvutsrqp of 2400 IUimg protein, was virtually free of filter paper activity, and had a ratio of contaminating filter paper activity of 2 x (0.009% endoglucanase activity). Approximately 3.3 g protein, which contained in excess of 7 x lo6 IU xylanase activity, was obtained from 17 L original culture filtrate. The process scheme was designed to facilitate scale-up to an industrial level of production. INTRODUCTION Lignocellulose, the world’s largest renewable bio- mass resource, is composed mainly of lignin, cellulose, and hemicellulose, of which a large part of the latter is xylan.’ Much of the current research and develop- ment effort has been directed toward the utilization of the cellulose fraction for liquid fuel production. How- ever, if value-added products could be obtained from the hemicellulose and lignin streams, the economics of the process could be significantly improved. For a number of years, our laboratory2 has been working on a process where aspenwood chips are first * To whom all correspondence should zyxwvutsrq be addressed; present ad- dress: Allelix, Inc., zyxwvutsrqpon 6850 Goreway Dr., Mississauga, Ontario L4V IP1. Canada. steam exploded to enhance their subsequent enzymatic hydrolysis to fermentable sugars. The hemicellulose, lignin, and cellulose streams can then be separated by selectively extracting the pretreated material by water and dilute alkali. The cellulose and hemicellulose streams are then, respectively, hydrolyzed by cellulases and xylanases and fermented to ethanol and 2,3-butanediol by the appropriate microorganisms. Previously, we had shown that high levels of cel- lulase and xylanase were efficiently produced by the fungus Triclioderma harzianum E58.3 As the hemicel- lulose- and cellulose-derived sugars are normally uti- lized by different microorganisms for various products, the hemicellulose and cellulose fractions are routinely separated into different streams in our process.2 Ide- ally, if an inexpensive process is available for the sep- aration of the xylanase complex from the cellulase complex, the two enzyme streams could be efficiently utilized for the hydrolyses of the hemicellulose and cellulose streams, respectively, while reducing the overall cost of separate enzyme production steps. A purified cellulase-free xylanase could also be used for the removal of contaminating hemicellulose com- ponents from high-grade cellulose pulps. Other workers4 have been partially successful at reducing the amount of hemicellulose in aspen mechanical pulps using pu- rified xylanase from Schizophyllum commune. Unfor- tunately, the viscosity of the pulp was also reduced, possibly because of the relatively high concentration zy (-3%) of contaminating endoglucanase activity that was present in their preparation. In addition, the cost of producing the xylanase may be high since their pro- cess depended on fractional precipitation using a vol- ume ratio of 3: 1 ethanol-culture filtrate. An inexpen- sive process that produces virtually cellulase-free xylanase may be more amenable to this application. Other potential uses of xylanasesS include processes for the manufacture of liquid coffee, the adjustment of Biotechnology and Bioengineering, Vol. XXX, Pp. 96-100 (1987) zyxwvuts 0 1987 ..John Wilev 81 Sons, Inc. CCC 0006-3592/87/’010096-05$04.00