Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum.
Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli-Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium.
Department of Genetic Engineering, Hallym University, Kangwon-Do, Chuncheon, 200-702, Korea.
Seong Lyul
SL
Rhim
Myeong Soo
MS
Park
Geun Eog
GE
Ji
D000595:Amino Acid Sequence; D000681:Amylases / Q000096:biosynthesis* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000378:metabolism; D001644:Bifidobacterium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000681:Amylases