Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6

Abstract

Paenibacillus curdlanolyticus B-6 showed effective degradation activities for xylan and cellulose and produced an extracellular multienzyme complex (approximately 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 functional domains which belonged to glycosyl hydrolase family-11 and to carbohydrate-binding module family-36, respectively, 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.

Culture-Independent Phylogenetic Analysis of the Microbial Community in Industrial Sugarcane Bagasse Feedstock Piles

“…28) Lignocellulolytic bacteria of the classes Bacilli and Actinobacteria have been reported in soils and waste sites with high lignocellulose content, 29,30) and their synergism in the degradation of sugarcane cellulose has been reported. 31) In this study, several potential lignocellulose-degrading aerobic bacteria including ones from genera Alicyclobacillus, Paenibacillus, and Thermomonospora and a lignocellulolytic species, Acidiothermus cellulolyticus capable of producing a variety of extracellular and cellulosomal-lignocellulose degrading enzyme systems, [32][33][34][35] could play a concerted role in bagasse decomposition. In addition, Bacteroidetes and Acidobacteria could also contribute significantly to lignocellulose degradation in the ecosystem.…”

Paenibacillus curdlanolyticus Strain B-6 Multienzyme Complex: A Novel System for Biomass Utilization

“…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.…”

“…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.…”

“…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.…”

“…To understand the xylanase system, a genomic library of the strain B-6 was constructed and screened for high xylanase activity. Recently, six xylanase genes, S1 (Pason et al, 2010), xyn10A (Waeonukul et al, 2009a), xyn10B (Sudo et al, 2010), xyn10C (unpublished data), xyn10D (Sakka et al, 2011) and xyn11A (Pason et al, 2010) were cloned, and the translated products were characterized (Table 4). …”