Novobiocin-Sepharose was prepared by coupling of novobiocin to Epoxy-activated Sepharose 6B and used as an affinity adsorbent. Four novobiocin-binding proteins were isolated from crude extracts of Escherichia coli with molecular weights of 105, 92, 85 and 40 kdal. The two larger proteins were identified as the A subunit (gyrA protein) and the B subunit (gyrB protein) of DNA gyrase topoisomerase II). By this method the two gyrase components can be easily separated and purified in high yield. Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP. The gyrB protein ATPase activity is competitively inhibited by novobiocin and related coumarin antibiotics. ATP hydrolysis is unaffected by the addition of either gyrA protein or DNA but stimulated in the presence of both.

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The nucleotide sequence of the celZ gene coding for a thermostable endo-beta-1,4-glucanase (Avicelase I) of Clostridium stercorarium was determined. The structural gene consists of an open reading frame of 2958 bp which encodes a preprotein of 986 amino acids with an Mr of 109,000. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase I purified from C. stercorarium culture supernatants. The recombinant protein expressed in Escherichia coli is proteolytically cleaved into catalytic and cellulose-binding fragments of about 50 kDa each. Sequence comparison revealed that the N-terminal half of Avicelase I is closely related to avocado (Persea americana) cellulase. Homology is also observed with Clostridium thermocellum endoglucanase D and Pseudomonas fluorescens cellulase. The cellulose-binding region was located in the C-terminal half of Avicelase I. It consists of a reiterated domain of 88 amino acids flanked by a repeated sequence about 140 amino acids in length. The C-terminal flanking sequence is highly homologous to the non-catalytic domain of Bacillus subtilis endoglucanase and Caldocellum saccharolyticum endoglucanase B. It is proposed that the enhanced cellulolytic activity of Avicelase I is due to the presence of multiple cellulose-binding sites.

Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium. A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9. Determination of the N-terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I. Maximal activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75 degrees C. Hydrolysis of avicel occurs at a linear rate for three days at 70 degrees C. Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins. It lacks activity towards carboxymethylcellulose and barley beta-glucan. Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl-beta-D-cellobiosides. Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1. With acid-swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose. The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non-reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non-reducing end of the cellulose chains.

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...