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

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons78335

Lottspeich,  F.
Lottspeich, Friedrich / Protein Analysis, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Fuchs, K. P., Zverlov, V. V., Velikodvorskaya, G. A., Lottspeich, F., & Schwarz, W. H. (2003). Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-beta-1, 3-glucanase bound to the outer cell surface. Microbiology-SGM, 149, 1021-1031.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-6C43-A
Abstract
Clostridium thermocellum produces one major beta-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-beta- 1,3-1,4-glucanase CelC, a putative transcription regulator of the Lacl type, beta-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 as 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)-beta- 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 beta-glucan and laminarin, respectively. It was specific for beta-glucans containing beta-1,3-linkages with an optimum temperature of 70degreesC 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 beta- 1,3- or beta-1,3-1,4-glucan.