RRC ID 3339
著者 Tamura H, Yamada A, Kato H.
タイトル Identification and characterization of a dextranase gene of Streptococcus criceti.
ジャーナル Microbiol Immunol
Abstract 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.
巻・号 51(8)
ページ 721-32
公開日 2007-1-1
DOI 10.1111/j.1348-0421.2007.tb03961.x
PII JST.JSTAGE/mandi/51.721
PMID 17704634
MeSH Amino Acid Sequence Base Sequence Cloning, Molecular Dextranase / genetics* Dextranase / metabolism Dextrans / chemistry Electrophoresis, Polyacrylamide Gel Molecular Sequence Data Mutagenesis, Site-Directed Phylogeny Polymerase Chain Reaction Streptococcus / enzymology* Streptococcus / genetics
IF 1.566
引用数 4
WOS 分野 IMMUNOLOGY MICROBIOLOGY
リソース情報
原核生物(大腸菌) CV-25(DH5alpha)