Identification and characterization of a dextranase gene of Streptococcus criceti.
The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene-walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200-amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell-surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C-terminus was partially purified and showed dextranase activity on blue-dextran sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDSPAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD-SDS-PAGE, whereas no dextranase activity of the cells was observed on blue-dextran brain heart infusion (BD-BHI) agar plates. Furthermore, PCR-based mutations of dextranase indicated that a deletion mutant of the C-terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C-terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp-453 and Trp-793 residues of S. criceti dextranase are critical to the enzyme's activity.
Tamura
Haruki
H
Department of Dental Pharmacology, School of Dentistry, Iwate Medical University, Morioka, Iwate 020-8505, Japan. htamura@iwate-med.ac.jp
Yamada
Arisa
A
Kato
Hirohisa
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003910:Dextranase / Q000235:genetics* / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics
D003911:Dextrans; C017080:blue dextran; D003910:Dextranase