Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module

Abstract

The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548-554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTDeltadoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTDeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan, and low activity toward xylan. The V(max) and K(m) values were 137 micro mol min(-1) mg(-1) and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

Enzymatic diversity of the Clostridium thermocellum cellulosome is crucial for the degradation of crystalline cellulose and plant biomass

“…To reconstitute the C. thermocellum cellulosome, we selected 40 type-I dockerin-containing proteins that were previously identified by proteomic analysis as components of the native cellulosome isolated from cells grown on crystalline cellulose 7 ( Fig. 1 ): 23 cellulases, which include 4 exoglucanases (Cel48S 15 , Cel9K 16 , Cbh9A 17 , and Cel5O 18 ) and 19 endoglucanases (Cel8A 19 , Cel9Q 20 , Cel9F 21 , Cel5B 22 , Cel9T 23 , Cel9R 24 , Cel5G 25 , Cel5E 26 , Cel9,44J 27 , Cel9W, Cel9P, Cel9N 28 , Cel5L, Cel9D 29 , Cel9V, Cel5,26H 30 , Cel124A 31 , GH9 (Cthe_2761), and GH9 (Cthe_0433)); 10 hemicellulases, which include six xylanases (Xyn11A 32 , Xyn10C 33 , Xyn10Z 34 , Xyn10Y 35 , Xyn5A 36 , and Xyn30A 37 ), three mannanases (Man5A 38 , Man26A 39 , and Man26B 40 ), and one xyloglucanase (Xgh74A 41 ); four pectic enzymes, which include two galactanases (GH53 (Cthe_1400) and Gal43A 42 ), one rhamnogalacturonan lyase (Rgl11A 43 ), and one rhamnogalacturonan acetyl esterase (Rgae12A); and three other proteins, which include one lichenase (Lic16B 44 ), one chitinase (Chi18A 45 ), and one protease inhibitor (serpin 46 ). Proteins with unknown functions previously identified as cellulosomal components were not used in this study.…”

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

“…The hydrolytic activity of the enzymes (0.5 M) against linear arabino-oligosaccharides (10 mg/ml; degree of polymerization, 2 to 6) in citrate buffer (pH 5.5, 50 mM; 50 mM NaCl; 15 h of incubation) was analyzed by thin-layer chromatography and HPAEC-PAD as described previously (59)(60)(61)(62). Branched arabino-oligosaccharides (100 M) were incubated with the enzymes (final concentration, 1 M) for 16 h at 60°C.…”

Impact of Pretreated Switchgrass and Biomass Carbohydrates on Clostridium thermocellum ATCC 27405 Cellulosome Composition: A Quantitative Proteomic Analysis

“…Some of the catalytic components possessed diverse hydrolytic activities ranging from exoglucanases (CelS, CbhA, CelK) to endoglucanases (CelA, CelF, CelN, CelB), xylanases (XynA/U, XynY, XynZ), and other hemicellulases (LicB, ChiA), and some proteins contained multiple catalytic modules (CelH, CelJ). [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26].…”