CLONING AND DNA-SEQUENCING OF BGAA, A GENE ENCODING AN ENDO-BETA-1,3-1,4-GLUCANASE, FROM AN ALKALOPHILIC BACILLUS STRAIN (N137)

The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichen ase) from an alkalophilic Bacillus sp. strain N137, isolated in our la boratory, was cloned and expressed from its own promoter in Escherichi a coli. The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleoti des. The deduced protein sequence consists of 276 amino acids and has a 31-amino-acid putative signal peptide which is functional in E. coli , in which the BgaA protein is located mainly in the periplasmic space . The lichenase activity of BgaA is stable between pH 6 and 12, it sho ws optimal activity at a temperature between 60 and 70-degrees-C, and it retains 65% of its activity after incubation at 70-degrees-C for 1 h. This protein is similar to some other lichenases from Bacillus spec ies such as B. amyloliquefaciens, B. brevis, B. licheniformis, B. mace rans, B. polymyxa, and B. subtilis. However, it has a lysine-rich regi on at the carboxy terminus which is not found in any other published l ichenase sequence and might be implicated in the unusual biochemical p roperties of this enzyme. The location of the mRNA 5' end was determin ed by primer extension and corresponds to nucleotide 235. A typical Ba cillus sigma(A) promoter precedes the transcription start site.