Characterization and structure of the cellulase gene of Bacillus subtilis BSE616.

Abstract

The Bacillus subtilis carboxymethyl cellulase (CMCase) gene originally cloned on a 3.2-kb PstI DNA fragment has been localized in a 1.5-kb Sau3AI fragment by a series of subclonings into plasmid pUC19. During the process the promoter region and Shine-Dalgarno (SD) sequence were deleted, but the 1.5-kb insert was shown to direct the synthesis of CMCase in scherichia coli to a high level, probably with the aid of lac promoter. We analyzed the complete nucleotide sequence of the CMCase gene. The CMCase gene is 1500-bp long, encoding a polypeptide of 499 amino acids and a stop codon. The putative "-35" region (TAGACA), "-10" region (TACAAT), and ribosome binding site (RBS) (AAGGAGG) have also been identified in the 5' flanking region. Comparison of the nucleotide sequence to three other published endo-beta-1,4-glucanase genes of B. subtilis strains shows that these sequences share very strong homology. It seems that the cellulase genes have been derived from a common ancestor by spontaneous mutation. The probability of carboxy-terminal processing of the CMCase protein is also discussed.