Roy H. Doi

Cellulosomes: plant-cell-wall-degrading enzyme complexes

Cellulose, the main structural component of plant cell walls, is the most abundant carbohydrate polymer in nature. Although abundant, it is extremely difficult to degrade, as it is insoluble and is present as hydrogen-bonded crystalline fibres. Anaerobic microorganisms have evolved a system to break down plant cell walls that involves the formation of a large extracellular enzyme complex called the cellulosome, which consists of a scaffolding protein and many bound cellulases. Cellulosomes have many potential biotechnological applications as the conversion of cellulosic biomass into sugars by cellulosomes could result in the production of high-value products such as ethanol or organic acids from inexpensive renewable resources. Rapid advances in cellulosome research are providing basic information for the development of both in vitro and in vivo systems to achieve such goals.

Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases from Clostridium cellulovorans

Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.

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

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.

Degradation of Corn Fiber by Clostridium cellulovorans Cellulases and Hemicellulases and Contribution of Scaffolding Protein CbpA

ABSTRACT Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.

Xylanase and Acetyl Xylan Esterase Activities of XynA, a Key Subunit of the Clostridium cellulovorans Cellulosome for Xylan Degradation

ABSTRACT The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-β-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans.

Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome

Clostridium cellulovorans uses not only cellulose but also xylan, mannan, pectin, and several other carbon sources for its growth and produces an extracellular multienzyme complex called the cellulosome, which is involved in plant cell wall degradation. Here we report a gene for a cellulosomal subunit, pectate lyase A (PelA), lying downstream of the engY gene, which codes for cellulosomal enzyme EngY. pelA is composed of an ORF of 2,742 bp and encodes a protein of 914 aa with a molecular weight of 94,458. The amino acid sequence derived from pelA revealed a multidomain structure, i.e., an N-terminal domain partially homologous to the C terminus of PelB of Erwinia chrysanthemi belonging to family 1 of pectate lyases, a putative cellulose-binding domain, a catalytic domain homologous to PelL and PelX of E. chrysanthemi that belongs to family 4 of pectate lyases, and a duplicated sequence (or dockerin) at the C terminus that is highly conserved in enzymatic subunits of the C. cellulovorans cellulosome. The recombinant truncated enzyme cleaved polygalacturonic acid to digalacturonic acid (G2) and trigalacturonic acid (G3) but did not act on G2 and G3. There have been no reports available to date on pectate lyase genes from Clostridia.

Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800.

ABSTRACT Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B. subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.

Characterization of Two Noncellulosomal Subunits, ArfA and BgaA, from Clostridium cellulovorans That Cooperate with the Cellulosome in Plant Cell Wall Degradation

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.

Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates

Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized. Most of the activity was secreted into the growth medium when the bacterium was grown on xylan. Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions. Each of these fractions contained at least two major and three minor xylanase activities. In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis. The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000. High alpha-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions. Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan. These results strongly indicated that C. cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate.

Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation

The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon “intercellular complementation.” Interesting implications concerning bacterial cooperation are suggested from these results.