
Cloning and sequencing of a cyclodextrin glycosyltransferase gene from Brevibacillus brevis CD162 and its expression in Escherichia coli
Author(s) -
Kim Myung Hee,
Sohn Cheon Bae,
Oh Tae Kwang
Publication year - 1998
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1998.tb13117.x
Subject(s) - puc19 , escherichia coli , biology , biochemistry , nucleic acid sequence , peptide sequence , microbiology and biotechnology , recombinant dna , molecular cloning , open reading frame , gene
A cyclodextrin glycosyltransferase (CGTase) gene of Brevibacillus brevis CD162 was cloned into Escherichia coli using pUC19 as a vector. Determination of the nucleotide sequence showed the presence of an open reading frame of 2079 bp encoding a polypeptide of 693 amino acid residues, composed of a 20‐amino acid signal sequence and a 673‐amino acid mature enzyme. Neither a TATA‐ nor a TTGA‐like sequence was observed within the cloned DNA fragment. However, the fragment was expressed in Escherichia coli by the lac promoter of pUC19 and 74% of the total activity was secreted into the fermentation medium. The amino acid sequence of the mature CGTase showed the highest homology of 86% to that of Bacillus sp. KC201. The CGTase purified to homogeneity from the recombinant E. coli exhibited the same properties as those of native CGTase from Brevibacillus brevis CD162 in terms of molecular mass, reaction conditions, stability and the production of cyclodextrins.