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Pure Appl. Chem., Vol. 70, No. 11,
pp. 2124, 1998
Characterization of a novel rotenoid-b -glucosidase
enzyme and its natural substrate from Thai Rosewood*
M.R. Jisnuson Svasti1,2, Chantragan
Srisomsap1, Rudee Surarit1,3, Supanna Techasakul1,4,
and James Ketudat-Cairns1,5
1. Chulabhorn Research Institute, Bangkok, Thailand
2. Faculty of Science & 3. Faculty of Dentistry, Mahidol University,
Bangkok, Thailand
4. Faculty of Science, Kasetsart University, Bangkok, Thailand
5. Institute of Science, Suranaree University of Technology,
Nakorn Ratchasima, Thailand
Abstract: A b -glucosidase
enzyme was purified from the seeds of Thai Rosewood (Dalbergia
cochinchinensis Pierre) by ammonium sulfate fractionation, preparative
isoelectric focusing and Sephadex G-150 chromatography. The electrophoretically
homogeneous product had molecular weights of 66,000 in the denatured
state and 330,000 in the native state. p-Nitrophenyl-b
-D-glucoside (Km 5.4 mM; kcat 307 sec-1)
and p-NP-b -D-fucoside (Km
0.54 mM; kcat 151 sec-1) were hydrolyzed at
the same active site with pH optimum of 5.0. Inhibition was observed
with d -gluconolactone, HgCl2,
conduritol B epoxide and Tris. The presumed natural substrate, present
in the seed at 3.5% by weight, was purified by silica gel column
chromatography and reversed phase TLC, and its structure was determined
by NMR. and mass spectroscopy to be a rotenoid b
-glucoside. Despite its unusual specificity, sequence analysis indicated
that rotenoid b -glucosidase is clearly
homologous to other b -glucosidases
in its N-terminal sequence. Reversal of the hydrolytic action of
Thai Rosewood b -glucosidase by 50%
(w/w) D-glucose at 50° C gave disaccharides
and trisaccharides in 30% yield, with gentiobiose as the major product.
Novel hetero-oligosaccharides could be synthesized from mixtures
of D-glucose and D-fucose. Since rotenoids have insecticidal properties,
the rotenoid glucoside and its specific b
-glucosidase enzyme may play a role in protection against insects.
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* Invited lecture presented at the International
Conference on Bioversity and Bioresources: Conservation and
Utilization, 23-37 November 1997, Phuket, Thailand.
Page last modified 27 April 1999.
Copyright ©1997, 98, 99 International Union of Pure and Applied
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