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

¹ Research Group Microbial Biotechnology, Technical University of Muenchen, Am Hochanger 4, D-85350 Freising, Germany
² Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia
³ Max-Planck-Institute for Biochemistry, Martinsried, Germany

Correspondence
Wolfgang H. Schwarz
schwarz{at}mikro.biologie.tu-muenchen.de

Clostridium thermocellum produces one major -1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237^T (6848 bp) and F7 (9766 bp). Overlapping sequences were 99·9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo--1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, -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)--glucanase with a specific activity of 2680 and 340 U mg^-1 and a K_m of 0·94 and 2·1 mg ml^-1 towards barley -glucan and laminarin, respectively. It was specific for -glucans containing -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 -1,3- or -1,3-1,4-glucan.

The GenBank accession numbers for the sequences reported in this paper are AJ307315 and X89732.

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