BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6 Patthra Pason & Akihiko Kosugi & Rattiya Waeonukul & Chakrit Tachaapaikoon & Khanok Ratanakhanokchai & Takamitsu Arai & Yoshinori Murata & Jun Nakajima & Yutaka Mori Received: 6 May 2009 / Revised: 27 June 2009 / Accepted: 28 June 2009 / Published online: 14 July 2009 # Springer-Verlag 2009 Abstract Paenibacillus curdlanolyticus B-6 showed effec- tive degradation activities for xylan and cellulose and produced an extracellular multienzyme complex (approxi- mately 1,450 kDa) containing several xylanases and cellulases. To characterize the multienzyme complex, we purified the complex from culture supernatants by four kind of chromatography. The purified multienzyme complex was composed of a 280-kDa protein with xylanase activity, a 260-kDa protein that was a truncated form on the C- terminal side of the 280-kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and carboxymethyl cellulase activities. The 280-kDa protein resembled the scaffolding proteins of cellulosomes based on its migratory behavior in polyacrylamide gels and as a glycoprotein. Cloning of the 40-kDa major xylanase subunit named Xyn11A revealed that Xyn11A contained two func- tional domains which belonged to glycosyl hydrolase family- 11 and to carbohydrate-binding module family-36, respec- tively, and a glycine- and asparagine-rich linker. However, an amino acid sequence similar to a dockerin domain, which is crucial to cellulosome assembly, was not found in Xyn11A. These results suggest that the multienzyme complex produced by P. curdlanolyticus B-6 should assemble by a mechanism distinct from the cohesin-dockerin interactions known in cellulosomes. Keywords Paenibacillus curdlanolyticus . Xylanase . Cellulase . Multienzyme complex . Hemicellulase Introduction The use of cellulosic biomass as a renewable source of energy via breakdown to sugars that can then be converted to liquid fuel is of great interest. Plant biomass contains a complex mixture of polysaccharides such as cellulose, hemicellulose (xylan and galactomannan), pectic substances (galacturonan and arabinogalactan), and other polysaccharides (e.g., type II arabinogalactan and fucoxyloglucan; Aspinall 1980). The hemicellulose and pectin polysaccharides, as well as the aromatic polymer lignin, interact with the cellulose fibrils, creating a rigid structure strengthening the plant cell wall. Therefore, complete and rapid hydrolysis of these polysacchar- ides requires not only β-1,4-glycosidic chain-cleaving enzymes such as endo-β-1,4-glucanase, cellobiohydrolase, and β- glycosidase but also the cooperation of many enzymes such as xylanolytic enzymes and side chain-cleaving enzymes such as β-1,4-xylanase and α-L-arabinofuranosidase (Thomson 1993). Apparently, the production of the multienzyme complex may have a number of advantages for the effective hydrolysis of cellulosic substances by the optimization of Patthra Pason and Akihiko Kosugi contributed equally to this work. P. Pason : A. Kosugi : R. Waeonukul : T. Arai : Y. Murata : J. Nakajima : Y. Mori (*) Post-harvest Science and Technology Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan e-mail: ymori@affrc.go.jp P. Pason : C. Tachaapaikoon Pilot Plant Development and Training Institute, King Mongkut’ s University of Technology Thonburi, Bangkuntien, Bangkok 10150, Thailand R. Waeonukul : K. Ratanakhanokchai School of Bioresources and Technology, King Mongkut’ s University of Technology Thonburi, Bangkuntien, Bangkok 10150, Thailand Appl Microbiol Biotechnol (2010) 85:573–580 DOI 10.1007/s00253-009-2117-2