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The nucleotide sequence of the Clostridium thermocellum gene bglA, coding for the thermostable j-glucosidase A, has been determined. The coding region of 1344 bp was identified by comparison with the N-terminal amino acid squence of recombinant b-glucosidase A purified from Escherichiu coli. The deduced amino acid sequence corresponds to a protein of 51482 Da. The coding region is flanked by putative promoter and transcription terminator sequences. The protein is unrelated to P-glucosidase B of C . thermocellz4m, but has a high level of similarity with other bacterial P-glucosidases and phospho-P-glucosidases. Similarity is also observed with the Bgalactosidase of the archaebacterium Sulfolobus solfuturicus. Unexpectedly, it was found that human lactasephlorizin hydrolase contains three copies of a sequence closely related to C. thermocellum P-glucosidase A (up to 40% sequence identity). These diverse 8-glucosidases can therefore be grouped into an enzyme family (BGA) of common structural design. Sequence comparison by hydrophobic cluster analysis revealed that all BGA enzymes share a well conserved region which is homologous to the catalytic domain of the widely distributed cellulase family A. A distinctive feature of this domain is the sequence motif His-Asn-Glu-Pro in which the catalytic residues His and Glu are separated by 35 -55 amino acid residues. The cellulase family A and the fl-glucosidase family BGA might thus be considered as members of a protein super-family comprising j-glucanases and flglycosidases from all three primary kingdoms of living organisms.P-Glucosidases (P-D-glucoside glucohydrolases) catalyze the hydrolysis of P-glycosidic bonds between glucose and aryl, alkyl, or saccharide groups. Most P-glucosidases exhibit a low specificity in regard to the aglycon portion of the substrate [l]. Many enzymes are also nonspecific with respect to the C4 configuration of the sugar moiety and hydrolyze both P-glucosides and B-galactosides [2]. A particular case of low substrate specificity is the mammalian enzyme lactase/phlorizin hydrolase (LPH) which contains two distinct Pglycosidases (b-galactosidase and aryl-P-glucosidase) within one polypeptide chain [3, 41. On the other hand, specificity is usually very high with respect to phosphorylation at the C6 Enzymes. Restriction endonucleases BumHT, EcoRI, EcoRV, HindIII, KpnI, PstI, PvuII (EC 3.1.21.4); S1 nuclease (EC 3.1.30.1); E. coli exonuclease III (EC 3.1.11.2); T4 DNA ligase (EC 6.5.1.1); E. coli DNA polymerase (EC 2.7.7.7); P-D-ghcosidase (cellobiase) (EC 3.2.1.21); P-D-galactosidase (lactase) (EC 3.2.1.23); phlorizin hydrolase (EC 3.2.1.62); 6-phospho-P-~-galactosidase (EC 3.2.1.85); 6-phospho-P-D-glucosidase (EC 3.2.1.86); endoglucanase (cellulase) (EC 3.2.1.4); lysozyme (EC 3.2.1.17).Note. The novel nucleotide sequence data publishcd here have been deposited with the EMBL sequence data bank and are available under the accession number X60268. position of the sugar residue. Thus, the bacterial phospho-Pglucosidases associated with the phosphoe...

The nucleotide sequence of the bglB gene, coding for the thermostable beta-glucosidase B of Clostridium thermocellum was determined. The coding region of 2265 bp was identified by comparison with the N-terminal amino acid sequence of beta-glucosidase B purified from Escherichia coli. The derived amino acid sequence corresponding to a polypeptide of Mr 84,100 was confirmed by sequencing of the C-terminal peptide generated by cleavage with cyanogen bromide. The protein bears no resemblance to other bacterial beta-glucosidase sequences. However, extensive regions of homology were identified between the C. thermocellum enzyme and fungal beta-glucosidases. The N-terminal homologous region contains an amino acid sequence very similar to the active site of beta-glucosidase A3 from Aspergillus wentii. The striking sequence similarities between C. thermocellum beta-glucosidase B and Kluyveromyces fragilis beta-glucosidase suggest the possibility of a genetic exchange between thermophilic anaerobic bacteria and yeasts.

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A cellulase gene of Clostridium thermocellum was transferred to Escherichia coli by molecular cloning with bacteriophage lambda and plasmid vectors and shown to be identical with the celA gene. The celA gene product was purified from extracts of plasmid-bearing E. coli cells by heat treatment and chromatography on DEAE-Trisacryl. It was characterized as a thermophilic endo-4-1,4-glucanase, the properties of which closely resemble those of endoglucanase A previously isolated from C. thermocellum supernatants. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme purified from E. coli exhibited two protein bands with molecular weights of 49,000 and 52,000. It had a temperature optimum at 75°C and was stable for several hours at 60°C. Endoglucanase activity was optimal between pH 5.5 and 6.5. The enzyme was insensitive against end product inhibition by glucose and celiobiose and remarkably resistant to the denaturing effects of detergents and organic solvents. It was capable of degrading, in addition to cellulosic substrates, glucans with alternating 0-1,4 and 0-1,3 linkages such as barley I8-glucan and lichenan. The extracellular cellulase produced by the thermophilic anaerobic bacterium Clostridium thermocellum is attracting increased interest because of its potential for the saccharification of lignocellulosic biomass. It offers several advantages over mesophilic enzymes obtained from fungi of the genus Trichoderma. These include increased thermostability and a significantly higher specific activity (17, 26). Furthermore, in contrast to the fungal enzymes, the clostridial cellulase is not inhibited by moderate concentrations of the end products glucose and cellobiose. Cellulase is not a single enzyme but a multienzyme complex (6). It includes at least two types of enzymatic activities, namely, endoglucanases (1,4-p-D-glucan glucanohydrolases, EC 3.2.1.4) and exoglucanases (1,4-p-D-glucan