Sequence analysis,cloning and extracellular expression of cyclodextrin glucanotransferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in Escherichia coli

The cgt-gene from the alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was isolated and sequenced. An open reading frame (ORF) of 2112 bp encoding a polypeptide of 704 amino acids, composed of a 29-amino acid signal sequence and a 675-amino acid mature enzyme was found. The established low level of homology with nucleotide sequences of other Bacillus CGTases (less than 82%) suggested that the cgt-gene from Bacillus pseudalcaliphilus 8SB encodes a new enzyme. The cgt-gene was cloned as a PCR amplicon and thereby the construction of genome library was avoided. This is the first evidence for the use of pJET vector as an expression vector. The opportunity to apply its T7 promoter for efficient extracellular production of heterologous proteins in Escherichia coli BL21 (DE3) was demonstrated. The expression of extracellular recombinant CGTase improved 23-fold, concerning β-CGTase activity and 4.5-fold concerning γ-CGTase activity after IPTG induction and glycine supplementation was achieved.