Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis.
A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.
Okushima
M
M
Laboratory of Protein Engineering, Central Research Institute, Nissin Food Products Co., Ltd., Shiga.
Sugino
D
D
Kouno
Y
Y
Nakano
S
S
Miyahara
J
J
Toda
H
H
Kubo
S
S
Matsushiro
A
A
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003910:Dextranase / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D013298:Streptococcus sanguis / Q000235:genetics
D021382:Protein Sorting Signals; D003910:Dextranase