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To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. El, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTEl, was isolated. Nucleotide sequence analysis of the clone pCGTEl revealed that BCGTEl contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of β-cyclodextrin producer, Bacillus sp. KC201.
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