Isolation, characterization, and cloning of {alpha}-L-Arabinofuranosidase expressed during fruit ripening of Japanese pear.
alpha-L-Arabinofuranosidase (alpha-L-arafase) was purified from fruit of Japanese pear (Pyrus pyrifolia). The enzyme solubilized from the cell wall by NaCl and Triton X-100 had the homogeneity of a single 62-kD polypeptide on SDS-PAGE after purification through the steps of hydroxyapatite, anion-exchange chromatography, and size-exclusion chromatography. A related cDNA clone was isolated (PpARF2). The transcript and related protein were detected solely in the ripening fruit corresponding to the increase of alpha-L-arafase activity. Transcripts of PpARF2 were not detected in buds, leaves, roots, or shoots of the Japanese pear. The deduced amino acid sequences of PpARF2 had low identity with those of other plants or bacteria. This alpha-L-arafase belonged to glycoside hydrolase family 3, which includes some beta-xylosidases. The purified enzyme hydrolyzed mainly p-nitrophenyl alpha-L-arabinofuranoside and also reacted bifunctionally with p-nitrophenyl beta-d-xylopyranoside. However, it released only arabinose from native cell wall polysaccharides prepared from Japanese pear and from sugar beet arabinan. The enzyme did not release xylose from arabinoxylan and xylan. The only activity of the alpha-L-arafase presented here was hydrolyzing the arabinosyl residue from native polysaccharides, whereas it showed bifunctional activity against artificial substrates. According to the expression pattern and properties of the enzyme, it is a new member of the glycoside hydrolase family 3 isolated from fruit, and it may be responsible for modification of the cell wall architecture during fruit softening.
Tateishi
Akira
A
College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan. tateishi@brs.nihon-u.ac.jp
Mori
Hitoshi
H
Watari
Junya
J
Nagashima
Kenji
K
Yamaki
Shohei
S
Inoue
Hiroaki
H
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000201:enzymology; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007564:Japan; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010802:Phylogeny; D031989:Pyrus / Q000201:enzymology*; D013379:Substrate Specificity
D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase