Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137)

Abstract

The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp. strain N137, isolated in our laboratory, was cloned and expressed from its own promoter in Escherichia coli. The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleotides. 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 shows 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 species such as B. amyloliquefaciens, B. brevis, B. licheniformis, B. macerans, B. polymyxa, and B. subtilis. However, it has a lysine-rich region at the carboxy terminus which is not found in any other published lichenase sequence and might be implicated in the unusual biochemical properties of this enzyme. The location of the mRNA 5' end was determined by primer extension and corresponds to nucleotide 235. A typical Bacillus sigma A promoter precedes the transcription start site.