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

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)324-332
Number of pages9
JournalBioscience, Biotechnology and Biochemistry
Volume68
Issue number2
DOIs
Publication statusPublished - Feb 1 2004

Fingerprint

Chitinases
Mutagenesis
Chitin
Cloning
Molecular Cloning
Seed
Seeds
Complementary DNA
Substrates
Mutation
Trichoderma
Escherichia coli
Open Reading Frames
Amplification
Culture Media
Bacteria
Cytoplasm
Nucleotides
Amino Acids
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

Cite this

Molecular cloning, functional expression, and mutagenesis of cDNA encoding class I chitinase from rye (Secale cereale) seeds. / Ohnuma, Takayuki; Taira, Toki; Yamagami, Takeshi; Aso, Yoichi; Ishiguro, Masatsune.

In: Bioscience, Biotechnology and Biochemistry, Vol. 68, No. 2, 01.02.2004, p. 324-332.

Research output: Contribution to journalArticle

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