Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome

Abstract

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-␤-D-xylopyranoside, p-nitrophenyl-␤-D-cellobioside, p-nitrophenyl-␤-D-glucopyranoside, and carboxymethyl cellulose. The K m and V max values for xylan were 3.9 mg/ml and 611 mol/min/mg of protein, respectively. This enzyme was optimally active at 80°C and was stable up to 70°C at neutral pHs and over the pH range of 4 to 11 at 25°C.

Production and characterization of an enzyme complex from a new strain of Clostridium thermocellum with emphasis on its xylanase activity

“…The enzyme exhibited a slight activity against PNPX. In opposite to the present enzyme complex, XynC-II of cellulosome from C. thermocellum F1 and cellulosome-type enzymes of Bacteroides succinogenes S-85 showed β-glucosidase activity (7,8). In addition, the cellulosome of Bacteroides succinogenes S-85 consisted of eight endoglucanases and two xylanases.…”

“…The molecular mass values of X2, X3, X4 and X5 were estimated to be 97.4, 66, 48.5 and 34 kDa, respectively. X3 showed a molecular mass value close to xylanase XynC, one of the major component of C. thermocellum F1 cellulosome (8). The identification of the proteins responsible for cellulase and mannanase activities will help to establish if these activities are displayed by the same or different enzymes.…”

Cloning and Characterization of a β-1,4-Mannanase 5C Possessing a Family 27 Carbohydrate-Binding Module from a Marine Bacterium,Vibriosp. Strain MA-138

“…31) Some xylan-binding modules classified in family 22 were originally identified as thermostabilizing modules, because their removal from thermophilic enzymes resulted in decreases in the thermostability and/or optimal temperature of the enzymes. [50][51][52] Removal of the N-terminal CBM27 of Man26 of C. saccharolyticus strain Rt8B.4 resulted in a decrease in the optimal temperature of the enzyme. 53) However, removal of the C-terminal CBM27 region from Man5C of strain MA-138 did not affect its optimal temperature (data not shown).…”

Cohesin-Dockerin Interactions within and between Clostridium josui and Clostridium thermocellum

“…PCR was performed with pMK-2 as the template, and the resultant PCR fragments were cloned between the NheI and SalI sites of pET-28a(ϩ), yielding pCoh1-Cj, Coh2-Cj, pCoh5-Cj, and pCoh6-Cj. The DNA regions encoding the dockerin domains of C. thermocellum Xyn11A (19) and Xyn10C (20) and C. josui Aga27A (21) and Cel8A (22) were amplified from pKS101, pKS103, pCj-Aga27A, and pUCJ2E, respectively, with different combinations of PCR primers containing an artificial NheI or SalI recognition sequence, i.e. Ct-Xyn11ADocF and Ct-Xyn11ADocR for the dockerin domain of Xyn11A, Ct-Xyn10CDocF and Ct-Xyn10CDocR for that of Xyn10C, Cj-AgaADocF and CjAgaADocR for that of Aga27A, and Cj-Cel8ADocF and Cj-Cel8ADocR for that of Cel8A (Fig.…”