DNA-SEQUENCES AND EXPRESSION IN STREPTOMYCES-LIVIDANS OF AN EXOGLUCANASE GENE AND AN ENDOGLUCANASE GENE FROM THERMOMONOSPORA-FUSCA

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca hav e been cloned in Escherichia coli, and their DNA sequences have been d etermined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transforman ts. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S . lividans than in E. coli. Thin-layer chromatography of digestion pro ducts showed that E1 digests cellotriose, cellotetraose, and cellopent aose to cellobiose and a trace of glucose. E4 is poor at degrading cel lotriose and cleaves cellopentaose to cellotetraose and glucose or cel lotriose and cellobiose. It readily cleaves cellotetraose to cellobios e. E1 shows 59% identity to Cellulomonas fimi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cell ulase family E. Alignment of the amino acid sequences of Clostridium t hermocellum celD, E1, E4, and four other members of family E demonstra tes a clear relationship between their catalytic domains, although the re is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical m odification to be important for catalytic activity are conserved in al l seven proteins. The catalytic domains of E1 and E4 are not similar t o those of T. fusca E2 or E5, but all four enzymes share similar cellu lose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previous ly as the binding site for a protein that regulates induction.