Two new cellulosome components encoded downstream of celI in the genome of Clostridium thermocellum: the non-processive endoglucanase CelN and the possibly structural protein CseP

Abstract

Clostridium thermocellum produces a great number of extracellular cellulases which are free or cellulosome-bound. The nucleotide sequence of a gene cluster containing the genes celI, celN and cseP was determined from C. thermocellum strain F7. Gene products Cel9I and Cel9N are structurally related enzymes having a glycosyl hydrolase family 9 and a carbohydrate-binding module (CBM3c), but show characteristic differences: Cel9I is a non-cellulosomal protein with an additional CBM (CBM3b), whereas Cel9N contains a cellulosomal dockerin module and no additional CBM. Although Cel9I is a processive endoglucanase, Cel9N is non-processive. Both enzymes hydrolyse phosphoric acid swollen cellulose, but the products of hydrolysis are different. The CseP protein encoded in the gene cluster is the first component attached to the cellulosomal scaffoldin for which no catalytic activity could be detected. It was shown to be present in the cellulosome. Its sequence is homologous to the spore-coat assembly protein CotH of Bacillus subtilis, suggesting a structural role of CseP in the cellulosome.

Cellulase, Clostridia, and Ethanol

“…The first two operons found are in the cipA cluster as two tandem operons, including cipA plus olpB and orfp2 plus olpA ("Attachment of cellulosomes to the cell surface" above). More recently, a noncellulosomal endoglucanase, CelI, was shown to be clustered with cellulosomal cellulase CelN and a possible cellulosomal structural, component CseP (for cellulosomal element protein) (373). cseP codes for a type I dockerin-containing protein and is in the opposite strand and likely transcribed independently from celI and celN.…”

“…CelF, the CelS equivalent in C. cellulolyticum, also displays significant activity in reducing the viscosity of a CMC solution (271). Some of the cloned endoglucanase genes are celA (26), celB (105), celC (298), celD (143,326), celE (113), celF (245), celG (188), celH (361), celI (101,123,373), celJ (encoding component S2 of the complex) (1,8), celM (162), celN (373), celQ (9), celT (172), and celX (113).…”

“…However, in some family 9 cellulases, their respective catalytic domains are immediately adjacent to a family IIIc CBD, of which many aromatic amino acid residues on the planar strip thought to be crucial for binding to cellulose are not conserved (19). Examples include CelI (101,123,373), CelN (373), CelQ (9), CelF of C. thermocellum (19), CelG of C. cellulolyticum (90), EngH of C. cellulovorans (194,321), CelZ of C. stercorarium (134), and cellulase E4 of Thermomonospora fusca (132). Family IIIc CBDs are considered to play a very different role from that of the family IIIa CBDs.…”

Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes

“…Polymeric and oligomeric substrates were hydrolysed to completion under the conditions stated above. Hydrolysis products were separated on silica gel 60 plates, as described previously (Zverlov et al, 2003) or by HPLC (Beckmann System Gold, equipped with a deashing and a HPX-42A column, Bio-Rad) and refractometric sugar detection (ERC-7512, Erma).…”

Lignocellulases: a review of emerging and developing enzymes, systems, and practices

“…The extreme stabilities (e.g., pH and thermal) and multifunctional nature of enzymes produced by these cellulosome-expressing bacteria have revolved the attention of scientist on to understanding the structure and function of their genetic makeup in order to mimic the innate abilities. On account of the obvious benefits reported in the literature, scientists have consistently investigated the gene (Yagüe et al 1990;Zverlov et al 2003;Koeck et al 2013), fusion/modification of enzymes (Ciolacu et al 2010;Lee et al 2010;Ye et al 2010;Lee et al 2011;Nakashima et al 2014), and the optimal growth (Islam et al 2013;Reed et al 2014) of these useful microbes to harness their inherent benefits. For example, the biochemical and biophysical characteristics of multimodular enzymes from Clostridium thermocellum (Zverlov et al 2005;Tachaapaikoon et al 2012;Brunecky et al 2012;Hirano et al 2013;Yuan et al 2015) and Thermotoga maritima (Chhabra et al 2002;Carvalho et al 2004;Pereira et al 2010;Wu et al 2011) have been reported.…”