Molecular cloning, functional expression, and mutagenesis of cDNA encoding class I chitinase from rye (Secale cereale) seeds

Takayuki Ohnuma, Toki Taira, Takeshi Yamagami, Yoichi Aso, Masatsune Ishiguro

研究成果: Contribution to journalArticle査読

14 被引用数 (Scopus)

抄録

A cDNA encoding rye seed chitinase-a (RSC-a) was cloned by rapid amplification of cDNA ends and PCR procedures. It consists of 1,191 nucleotides and encodes an open reading frame of 321 amino acid residues. Recombinant RSC-a (rRSC-a) was produced in the oxidative cytoplasm of Escherichia coli Origami(DE3) in a soluble form by inducing bacteria at a low temperature (2o°C). Purified rRSC-a showed properties similar to the original enzyme from rye seeds in terms of chitinase activity toward a soluble substrate, glycolchitin, and an insoluble substrate, chitin beads, in chitin-binding ability to chitin, and in antifungal activity against Trichoderma sp. in vitro. rRSC-a mutants were subsequently produced and purified by the same procedures as those for rRSC-a. Mutation of Trp23 to Ala decreased the chitinase activity toward both substrates and impaired the chitin-binding ability. Furthermore, the antifungal activity of this mutant was weakened with increasing of the NaCl concentration in the culture medium. Complete abolishment of both activities was observed upon the mutation of Glu126 to Gln. The roles of these residues in both activities are discussed.

本文言語英語
ページ(範囲)324-332
ページ数9
ジャーナルBioscience, Biotechnology and Biochemistry
68
2
DOI
出版ステータス出版済み - 2 2004

All Science Journal Classification (ASJC) codes

  • バイオテクノロジー
  • 分析化学
  • 生化学
  • 応用微生物学とバイオテクノロジー
  • 分子生物学
  • 有機化学

フィンガープリント

「Molecular cloning, functional expression, and mutagenesis of cDNA encoding class I chitinase from rye (Secale cereale) seeds」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル