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Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation.

ABSTRACT: Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases. In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from Clostridium cellulovorans. XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose. The mixture of XynA and cellulases at a molar ratio of 1:2 showed the highest synergistic effect of 1.6 on corn cell wall degradation. The amounts both of xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of XynA and cellulases. Although synergistic effects on corn cell wall degradation were found in simultaneous reactions with XynA and cellulases, no synergistic effects were observed in sequential reactions. The possible mechanism of synergism between XynA and cellulases is discussed.

SUBMITTER: Murashima K

PROVIDER: S-EPMC148067 | biostudies-literature | 2003 Mar

REPOSITORIES: biostudies-literature

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Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation.

Murashima Koichiro K Kosugi Akihiko A Doi Roy H RH

Journal of bacteriology 20030301 5

Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases. In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from Clostridium cellulovorans. XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but ...[more]

PMID: 12591868

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Project description:BackgroundClostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation.ResultsWe cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant.ConclusionIn this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

Project description:Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the alpha-L-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with alpha-L-arabinofuranosidase or alpha-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to alpha-L-arabinofuranosidase and beta-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl alpha-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl alpha-L-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl alpha-L-arabinopyranoside but also p-nitrophenyl beta-D-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl alpha-L-arabinopyranoside and p-nitrophenyl beta-D-galactopyranoside were compared, the K(m) values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for alpha-L-arabinopyranose residues than for beta-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 beta-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with beta-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.

Dataset Information

Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation.

Publications

Synergistic effects of cellulosomal xylanase and cellulases from Clostridium cellulovorans on plant cell wall degradation.

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