Cloning and sequencing of a cyclodextrin glycosyltransferase gene from Brevibacillus brevis CD162 and its expression in Escherichia coli.
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.
Department of Food and Nutrition, Chungnam National University, Yusung, Taejon, South Korea.
M H
MH
Kim
C B
CB
Sohn
T K
TK
Oh
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase