Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

Abstract

Clostridium thermocellum produces one major b-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237T (6848 bp) and F7 (9766 bp).Overlapping sequences were 99?9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-b-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, b-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 aa with a calculated molecular mass of 148 kDa. Lic16A is an unusually complex protein consisting of a leader peptide, a threefold repeat of an S-layer homologous module (SLH), an unknown module, a catalytic module of glycosyl hydrolase family 16 and a fourfold repeat of a carbohydrate-binding module of family CBM4a. The recombinant Lic16A protein was characterized as an endo-1,3(4)-b-glucanase with a specific activity of 2680 and 340 U mg 21 and a K m of 0?94 and 2?1 mg ml 21 towards barley b-glucan and laminarin, respectively. It was specific for b-glucans containing b-1,3-linkages with an optimum temperature of 70˚C at pH 6?0. The N-terminal SLH modules were cleaved from the protein as well in Escherichia coli as in C. thermocellum, but nevertheless bound tightly to the rest of the protein. Lic16A was located on the cell surface from which it could be purified after fractionated solubilization. Its inducible production allowed C. thermocellum to grow on b-1,3-or b-1,3-1,4-glucan.

Cellodextrin and Laminaribiose ABC Transporters in Clostridium thermocellum

“…However, the binding stoichiometry ratios for glucose and cellobiose were higher than 1 (1.8 and 2.2, respectively). Since CbpC mocellum can also grow on laminarin (␤-1,3 glucan) as a sole carbon source (15), consistent with the presence of the observed laminaribiose transport system. Recently, laminaribiose was found to be the molecular inducer of the celC operon in C. thermocellum (22), which is composed of three genes, namely, celC (endo-␤-1,4-glucanase), licA [endo-␤-1,3(4)-glucanase], and glyR3 (celC operon repressor).…”

Mutations in the Scaffoldin Gene,cipA, ofClostridium thermocellumwith Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS1447, and Implications for Cellulase Synergism on Crystalline Cellulose

“…It was discussed as a possible cause for the "module shuffling" expected for cellulosomal genes and has been proposed to be the source of their modular composition. Multiple copies of this putative IS element were identified in the genome (10). However, no obvious connection between the locations of the cellulosomal genes and those of the IS1447 elements was found (data not shown).…”

Cellulase, Clostridia, and Ethanol

“…Another gene cluster consists of the genes encoding the noncellulosomal endoglucanase CelC, a LacI-like protein, the lichenan-degrading enzyme LicA, and a potential membrane protein with eight putative transmembrane regions (86). We (246) reported that this gene cluster is immediately upstream of the genes encoding ManB (171) and CelT (172).…”

“…It contains no dockerin domain but does have SLH segments (32). Similarly, LicA contains three SLH domains at its N-terminal end that have been shown to mediate its association with cells (86). Many scaffoldins, including that of C. cellulovorans (CbpA), do not have a dockerin.…”

“…The family 4 CBD of the C. thermocellum CelK has a binding capacity of ϳ4 mmol/g of cellulose (154), similar to those of the family 3 CBDs which have been characterized. It is interesting that LicA, a noncellulosomal C. thermocellum ␤-1,3-glucanase, has four family 4 CBDs at its C-terminal end (86).…”