Recently, the structures and mechanisms for assembly of multienzyme complexes, cellulosomes, in anaerobic cellulolytic microorganisms are clear (Bayer et al., 2004, 2007; Doi & Kosugi, 2004). Generally, the key feature of the cellulosome is a scaffoldin that integrates the various catalytic subunits into the complex by self-assembly by cohesion-dockerin interaction. However, the structure and mechanism of the multienzyme complex produced by a facultatively anaerobic bacterium, such as P. curdlanolyticus B-6 is still unknown. In order to describe features of the multienzyme complex system produced by strain B-6, the multienzyme complex was purified by four kinds of chromatography (cellulose affinity, gel filtration, anion-exchange and hydrophobic-interaction chromatographys) (Fig. 7).

The multienzyme complex of P. curdlanolyticus strain B-6 with molecular mass of 1,450 (G1) was isolated from culture supernatant at the late stationary growth phase through cellulose affinity and Sephacryl S-300 gel filtration chromatographys (Pason et al., 2006b). Basically, the individual cellulosomes from anaerobic bacteria show 600 kDa to 2.1 MDa complexes size and show cohesion-dockerin domain as a signature protein (Bayer et al., 2004; Doi & Kosugi, 2004). While, multienzyme complexes from aerobic microorganisms, were range in mass from about 468 kDa to 2 MDa (with contained 5-12 protein subunits) (Table 3) and has no report of cohesion-dockerin domain. Here, the multienzyme complex produced by strain B-6 under aerobic conditions was the first report on characterization.

Elucidation of the purified multienzyme feature of P. curdlanolyticus strain B-6 was followed by anion-exchange and hydrophobic-interaction chromatographys (Pason et al., 2010). The complex G1 from gel filtration chromatography (1,450 kDa) was purified by anion-exchange chromatography and showed at least five large protein complexes or aggregates, namely F1-F5. Among the fractions obtained from anion-exchange chromatography, F1 was apparently the most suited fraction to study on the organization and function of the multienzyme system of strain B-6 because F1 formed one clear band on the top of native PAGE, had the highest xylanase activity, and its subunit composition was clearly shown on SDS-PAGE. In the final step, complex F1 was separated to one major complex (H1) and two minor protein components (H2 and H3) by hydrophobic — interaction chromatography. The multienzyme complex (H1) was composed of a 280 kDa protein with xylanase activity, a 260 kDa protein that is 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 CMCase activities (Fig. 8). The two components (280 and 40 kDa) of the multienzyme complex has characteristics similar to the cellulosome of C. thermocellum in that it is composed of a scaffolding protein and a catalytic subunit (Bayer et al., 1998; Demain et al., 2005). The 280 kDa protein resembled the scaffolding proteins of the multienzyme complex based on its migratory behavior in polyacrylamide gels and as a glycoprotein. The 280 kDa protein and a 40 kDa major xylanase subunit are the key components of multienzyme complex of the strain B-

S1 protein: From the early research, the 280 kDa subunit (S1) plays a role of scaffoldin in assembling the enzyme complex and shows xylanase activity (Pason et al., 2010). The S1 gene consists of 2,589 nucleotides and encodes 863 amino acids with a molecular weight of 91,000 Da, indicating that the 280 kDa subunit is highly glycosylated. Sequence analysis revealed that S1 did not have significant homology with any proteins in the databases except for two surface layer homology (SLH) domains in its N-terminal region. Surprisingly, the recombinant S1 exhibits xylanase activity, and cellulose — and xylan-binding ability, suggesting that the S1 should be a novel xylanase and CBM(s) with new functions (unpublished data).

Xylanase Xyn10A: The xyn10A gene consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular weight of 142,726 Da. Xyn10A is a multidomain enzyme comprised of nine domains in the following order: three family-22 CBMs, a family-10 catalytic domain of glycosyl hydrolases (GH), a family-9 CBM, a glycine-rich region, and three SLH domains. Xyn10A can effectively hydrolyze insoluble xylan and natural biomass without pretreatment such as sugarcane bagasse, corn hull, rice bran, rice husk and rice straw. Xyn10A binds to various insoluble polysaccharides such as cellulose, xylan and chitin. The SLH domains functioned in Xyn10A by anchoring this enzyme to the cell surfaces of P. curdlanolyticus B-6. Removal of the CBMs from Xyn10A strongly reduced the ability of binding and plant cell wall hydrolysis. Therefore, the CBMs of Xyn10A play an important role in the hydrolysis of native biomass materials (Waeonukul et al., 2009a).

Xylanase Xyn10B: The xyn10B gene consists of 1,047 nucleotides encoding a protein of 349 amino acids with a predicted molecular weight of 40,480 Da. Xyn10B consists of only a family-10 catalytic of GH. Xyn10B is an intracellular endoxylanase (Sudo et al., 2010).

Xylanase Xyn10C: The xyn10C gene consists of 957 nucleotides and encodes 318 amino acid residues with a predicted molecular weight of 35,123 Da. Xyn10C is a single module enzyme consisting of a signal peptide and a family-10 catalytic module of GH (unpublished data).

Xylanase Xyn10D: The xyn10D gene consists of 1,734 nucleotides and encodes 577 amino acid residues with a calculated molecular weight of 61,811 Da. Xylanase Xyn10D is a modular enzyme consisting of a family-10 catalytic module of the GH, a fibronectin type-3 homology (Fn3) module, and family-3 CBM, in that order, from the N terminus. The CBM3 in Xyn10D has an affinity for cellulose and xylan, and plays an important role in hydrolysis of arabinoxylan and native biomass materials (Sakka et al., 2011).

Xylanase Xyn11A: The xyn11A gene consists of 1,150 bp and encodes a protein of 385 amino acids with a molecular weight of 40,000 Da. Xyn11A is composed of two major functional domains, a catalytic domain belonging to family-11 GH and a CBM classified as family-36. A glycine- and asparagine-repeated sequences existed between the two domains. Xyn11A has been identified to be one of the major xylanase subunit in the multienzyme complex of strain B-6 (Pason et al., 2010).

Based on both biochemical and molecular biological findings, a simplistic schematic view of the enzyme system from P. curdlanolyticus B-6 and its interaction with substrate and cell surface was created and presented in Fig. 9. In this assessment, the S1 protein did not have significant homology with any proteins in the databases except for two S-layer homology domains in its N-terminal region. However, the S1 protein that exhibits xylanase activity and cellulose — and xylan-binding ability, and contains cell anchoring function, seems remarkable. The multifunctional protein S1 is also responsible for forming the enzyme subunits into the complex and anchoring the complex into cell surface via the SLH domain. The interaction between S1 protein and enzyme subunits should be a mechanism distinct from the cohesion-dockerin interaction known in cellulosome of anaerobic microorganisms, since cohesion — or dockerin- like sequences were not observed in the S1 protein or the major xylanase subunit, Xyn11A. In addition, strain B-6 also produces cell bound multimodular xylanase Xyn10A that contains the numerous CBMs and SLH domains. Xyn10A can bind to the plant cell wall through CBM, whereas the catalytic module (GH10) is able to access its target substrate. Thus, the CBM greatly increases the concentration of the enzyme in the vicinity of the substrate, leading to the observed increase in polysaccharide hydrolysis. Besides, the presence of the functional CBMs and SLH domains in Xyn10A allows the cells to attach to substrate. Although, the overall structure of the enzyme complex system of the strain B-6 is not entirely clear, the enzyme complex has unique characteristics distinct from multienzyme complex cellulosome of anaerobic microorganisms. However, the mechanism for complex formation, interaction between the S1 protein as scaffoldin and enzyme subunits, needs to be further investigated.