For a resolution of reducing carbon dioxide emission and increasing food production to respond to the growth of global population, production of biofuels from non-edible biomass is urgently required. Abundant orange wastes, such as peel and strained lees, are produced as by-product of orange juice, which is available non-edible biomass. However, d-limonene included in citrus fruits often inhibits yeast growth and makes the ethanol fermentation difficult. This study demonstrated that isopropanol-butanol-ethanol fermentation ability of Clostridium beijerinckii and cellulosic biomass degrading ability of C. cellulovorans were cultivated under several concentrations of limonene. As a result, C. cellulovorans was able to grow even in the medium containing 0.05% limonene (v/v) and degraded 85% of total sugar from mandarin peel and strained lees without any pretreatments. More interestingly, C. beijerinckii produced 0.046 g butanol per 1 g of dried strained lees in the culture supernatant together with C. cellulovorans.

Plant cell wall-degrading enzymes have become increasingly important, since the development of efficient biomass degra- dation methods and the conversion of sugars to valuable prod- ucts such as butanol and amino acids and utilizable forms of energy such as ethanol and methane could lead to less depen- dence on imported petroleum as a fuel and chemical source. Plant biomass is an abundant renewable resource. Since cellu- lose and hemicellulose comprise about 40 to 50% of plant cell walls and are considered to be the largest components of the earth's biomass, efficient conversion of this material by engi- neered enzymes and/or microorganisms would be highly desir- able. The rate-limiting step in biomass degradation is the con- version of the cellulose and hemicellulose polymers to sugars. Basic research is essential to elucidate and improve the properties of enzymes that can degrade plant biomass, such as cellulose. Generally two types of cellulase systems occur. One type consists of independent extracellular cellulases that act synergistically to degrade cellulose, while the second type is represented by an enzyme complex called the "cellulosome," which consists of a nonenzymatic scaffolding protein associated with various enzymatic subunits that act in concert to degrade cellulose and hemicellulose. This minireview will focus on de- scribing the properties of cellulosomes from mesophilic bacte- ria and recent findings related to this interesting enzyme com- plex. The cellulosome from Clostridium thermocellum has been discussed in a number of recent reviews (2, 4, 5, 29). Reviews on cellulosomes (1, 3, 14, 15, 20, 57) and noncellulosomal cellulases (37, 48, 65) should be consulted for previous and complementary discussions of this subject. CELLULOSOMES What are cellulosomes? Cellulosomes are large extracellular enzyme complexes that are capable of degrading cellulose, hemicelluloses, and pectin and are produced by anaerobic bac- teria such as Clostridium, Acetivibrio, Bacteroides, and Rumino- coccus (Table 1). These microorganisms are found in various environmental niches, including soil, wood chip piles, sewage, and rumens. Cellulosomes actually function to degrade plant cell wall material and not only crystalline cellulose as its initial nomenclature suggested. Cellulosomes may be the largest ex- tracellular enzyme complexes found in nature, since polycellu- losomes have been reported to be as large as 100 MDa, al- though the individual cellulosomes range in mass from about 650,000 Da to 2.5 MDa. Cellulosomes were first observed by Bayer and Lamed as large protuberances on the surface of C. thermocellum (2, 31), and the complex was found to consist of a nonenzymatic scaf- folding protein to which were attached a number of enzymatic subunits (3). These observations on the complex structure were supported by the findings of Mayer et al. (38), who found by electron microscopy that cellulosomes had an ordered arrange- ment of subunits. There has been a relatively rapid expansion in identification of cellulosomes from various anaerobic cellu- lolytic bacteria (Table 1), and it is likely that cellulosomes will be identified in a large number of anaerobic bacteria. Cellulo- somes are now thought to degrade not only crystalline cellu- lose, but also hemicelluloses, chitin, and even pectin, depend- ing on the source of the cellulosomes. Interestingly, in one case, cellulosome-like structures were observed with Clostrid- ium acetobutylicum, but they did not have any activity in de- grading crystalline forms of cellulose (55). Recent studies have been devoted to characterizing and comparing the properties of the scaffolding protein and the cellulosomal enzymes from various bacterial sources. They have provided a glimpse into the diversity of cellulosomes and a partial rationale for their existence.

An anaerobic mesophile, Clostridium cellulovorans, produces a multienzyme complex called the cellulosome and actively degrades polysaccharides in the plant cell wall. C. cellulovorans also changes cellulosomal subunits to form highly active combinations dependent on the carbon substrate. A previous study reported on the synergistic effects of exoglucanase S (ExgS) and endoglucanase H (EngH) that are classified into the glycosyl hydrolase (GH) families 48, and 9, respectively. In this study, we investigated synergistic effects of ExgS and EngK, a GH9 cellulase different from EngH. In addition, since EngK was known to produce cellobiose as its main product, the inhibition on cellulase activity of EngK with cellobiose was examined. As a result, the effect of cellobiose inhibition on EngK coexistent with ExgS was found to be much lower than that with EngH. Thus, although EngH and EngK are in the same GH9 family, enzymatic activity in the presence of cellobiose was significantly different.

In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a “designer” cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli . The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.

A large gene cluster for the Clostridium cellulovorans cellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C. Liu and R. H. Doi, Gene 211:39–47, 1998). Gene walking revealed that the engL gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244–247, 2000) was located downstream of the cbpA-exgS genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of the clustered genes is cbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN and is about 22 kb in length. The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene. This large gene cluster is flanked at the 5′ end by a putative noncellulosomal operon consisting of nifV-orf1-sigX-regA and at the 3′ end by noncellulosomal genes with homology to transposase ( trp ) and malate permease ( mle ). Since gene clusters for the cellulosome are also found in C. cellulolyticum and C. josui , they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.

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 .