Clostridium cellulovorans Cellulosome and Cellulases
Roy H. DOI*, Laercio M. MALBURG Jr., Yutaka TAMARU, Akihiko ICHI-ISHI,Chi- Chi LIU and Atef IBRAHIM
Department of Molecular & Cellular Biology, University of California, Davis,CA 95616, USA
The anaerobic spore-forming bacterium Clostridium cellulovorans is capable of rapidly digesting cellulose. The cellulase activity consists of the cellulosome and non- cellulosomal cellulases. The cellulosome is a complex structure consisting of a non- enzymatic scaffolding protein, CbpA, and a number of enzymatic subunits associated with the CbpA. The non-cellulosomal cellulases are formed in addition to the cellulosome and complement the activity of the cellulosome.
The cbpA gene for CbpA has been cloned and sequenced and from the derived amino acid sequence, several features are noted. The CbpA has a molecular weight of 189,000 and exhibits a number of different domains. There is a signal peptide sequence for secretion of CbpA, a cellulose binding domain (CBD) at the N-terminus of the protein, four conserved hydrophilic domains (HLD) and nine conserved hydrophobic domains (HBD). The role of the CBD domain (MW = 16,000) is to bind the cellulosome to the cellulose substrate, since the cellulosome-associated enzymes do not have a strong cellulose binding domain. The cellulosomal enzymes do contain a conserved duplicated sequence (DS) usually at their C-termini that allows them to bind tightly to the nine HBDs. The role of the HLDs is still uncertain, although they may play some spacer role in the CbpA.
Mini-CbpAs have been constructed consisting of the CBD, HLD, and one or more HBDs. These mini-CbpAs are capable of binding the cellulosomal enzymatic subunits and are capable of degrading acid swollen cellulose (ASC). These constructs indicate that the HBDs, which are quite homologous, may vary in their binding capacity to the various cellulosomal enzymes, since HBD 1 and HBD6 have different activities when they form mini-cellulosomes.
SDS-PAGE analysis of the cellulosome indicate that C. cellulovorans cellulosomes consist of three major subunits, the CbpA, P 100 and ExgS and a number of minor enzymatic subunits. The P 100 is a large enzyme (MW of about 100,000) which has endoglucanase activity. The ExgS (MW = 75,000) has exoglucanase activity. Several genes for minor cellulosomal enzyme subunits have been cloned and sequenced. All of them contain a DS region near their C-termini.
The growth of C. cellulovorans with different carbon sources indicate that the genes for cellulosomal and non-cellulosomal cellulase subunits are regulated. Growth of the cells in glucose or cellobiose reduces dramatically the expression of cellulosomal subunits while enhancing the production of several non-cellulosomal cellulase enzymes, notably EngF and another protein with endoglucanase activity with a MW of about 120,000. During growth on glucose or cellobiose, the subunits of the cellulosome are produced in very low quantities as free entities. Their association into cellulosomes is enhanced when cellulose is added to the cell-free mixture of cellulosomal subunits. Growth on cellulose on the other hand significantly increases the synthesis of the cellulosome. Non-cellulosomal enzymes and cellulosomes are produced simultaneously and presumably act synergistically to degrade cellulose under various conditions of growth.
