Isolation of novel alkaliphilic Bacillus strains for cyclodextrin glucanotransferase production

New alkaliphilic Bacillus producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) were isolated from 17 Bulgarian alkaline and normal habitats (springs and soils) by three steps of a selection. None of the isolates obtained, producing CGTase, appeared to be thermophilic in character. One hundred and thirty-seven strains were estimated for CGTase activity by batch cultivation in a liquid alkaline medium. Twenty-seven of them had a detectable CGTase activity in their culture supernatants under the enzyme assay conditions, despite of the significant growth of all isolates. The phenotypic properties of three selected strains (20RF, 8SB and 24WE) were determined. They were aerobic endospore-forming Bacillus strains: two of them were obligated alkaliphiles (20RF and 8SB) and one, alkalitolerant (24WE). Both obligated alkaliphiles were further characterised by 16S rRNA analysis. According to the full 16S rRNA gene sequences obtained and deposited to the NCBI GenBank database, both isolated obligated alkaliphiles 20RF and 8SB were clustered into the group of alkaliphilic Bacillus species. The exhibited CGTase production by them (230-250 U ml(-1) for 20RF and 130-160 U ml(-1) for 8SB) defined these new isolates as promising producers of the enzyme, especially Bacillus sp. 8SB synthesising thermostable alkaline beta-CGTase. Both new enzymes from 20RF and 8SB Bacillus strains formed only two types of cyclodextrins, beta and gamma, which could be of interest for their easy separation and industrial production.