Bioscience, Biotechnology and Biochemistry p. 934 - 939 (2013)
Update date:2022-08-11
Topics:
Himeno, Nami
Saburi, Wataru
Wakuta, Shinji
Takeda, Ryosuke
Matsuura, Hideyuki
Nabeta, Kensuke
Sansenya, Sompong
Ketudat Cairns, James R.
Mori, Haruhide
Imai, Ryozo
Matsui, Hirokazu
β-Glucosidases (EC 3.2.1.21) split β-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release β-D-glucose. One of the important functions of plant β-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonaterelated compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid β-D-glucoside in rice (Oryza sativa L.). The expression of OsTAGG2 is upregulated by wounding and by methyl jasmonate, suggesting that this isozyme is involved in responses to biotic stresses and wounding, but the physiological substrate of OsTAGG2 remains ambiguous. In this study, we produced recombinant OsTAGG2 in Pichia pastoris (rOsTAGG2P), and investigated its substrate specificity in detail. From 1L of culture medium, 2.1mg of purified recombinant enzyme was obtained by ammonium sulfate precipitation and Ni-chelating column chromatography. The specific activity of rOsTAGG2P (182 U/mg) was close to that of the native enzyme (171 U/mg), unlike recombinant OsTAGG2 produced in Escherichia coli, which had approximately 3-fold lower specific activity than the native enzyme. The optimum pH and temperature for rOsTAGG2P were pH 3.4 and 60 βC. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4-9.8 and below 55 βC. Native OsTAGG2 and rOsTAGG2P showed 4.5-4.7-fold higher activities towards salicylic acid β-D-glucoside, an inactive storageform of salicylic acid, than towards tuberonic acid β-Dglucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity.
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