Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli.
The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.
Mizuno
T
T
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
Mori
H
H
Ito
H
H
Matsui
H
H
Kimura
A
A
Chiba
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C091051:glucan 1,6-alpha-isomaltosidase