Isolation and characterization of an enzyme with beta-glucosidase and beta-fucosidase activities from Dalbergia cochinchinensis Pierre.
A glycosidase enzyme with both beta-glucosidase and beta-fucosidase activities has been purified from the seeds of Dalbergia cochinchinensis Pierre (Thai Rosewood) by ammonium sulfate fractionation, preparative isoelectric focusing, and Sephadex G-150 chromatography. The enzyme has molecular weights of 330,000 in the native state and 66,000 in the denatured state. Hydrolysis of p-NP-beta-D-glucoside and p-NP-beta-D -fucoside showed pH optimum at pH 5.0 and was inhibited by delta-gluconolactone, HgCl2, and p-chloromercuribenzoate. The Km and kcat values of the purified enzyme were 5.4 mM and 307 s-1 for p-NP-beta-D-glucoside and 0.54 mM and 151 s-1 for p-NP-beta-D-fucoside, so that the latter had by far the higher kcat/Km ratio. p-NP-beta-D-galactoside, p-NP-beta-D-xyloside, and p-NP-alpha-L-arabinoside were hydrolyzed more slowly. Hydrolysis of sophorose, laminaribiose, and gentiobiose were also rather slow, and hydrolysis of cellobiose was even slower. No hydrolysis of the cyanogenic glucosides linamarin or prunasin, but some hydrolysis of amygdalin and salicin was found. Further studies are required to identify the natural substrates of the enzyme. However, high yields, ease of purification, and storage stability of the enzyme make it a useful candidate for various applications, such as study of oligosaccharide synthesis by reversal of hydrolysis.
Srisomsap
C
C
Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand.
Svasti
J
J
Surarit
R
R
Champattanachai
V
V
Sawangareetrakul
P
P
Boonpuan
K
K
Subhasitanont
P
P
Chokchaichamnankit
D
D
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000302:isolation & purification* / Q000378:metabolism; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
D001617:beta-Glucosidase; C028219:beta-D-fucosidase; D005644:alpha-L-Fucosidase