Purification and characterization of an isoflavone-conjugates-hydrolyzing beta-glucosidase from endophytic bacterium.
An isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) from endophytic bacterium, Pseudomonas ZD-8 was purified to homogeneity by successive ammonium sulfate precipitation, gel filtration on SephadexG-100, DEAE-sephrose CL-6B and DEAE-Sephacel chromatography. The enzyme was a monomeric protein with an apparent molecular mass of 33 kDa as determined by SDS-PAGE and gel filtration. It was optimally active at pH 6.0 and 40 degrees C and had a specific activity of 1485 U mg of protein(-1) against genistin. The ICHG readily hydrolyzed rho-nitrophenyl-beta-glucoside, rho-nitrophenyl-beta-galactoside, genistin, daidzin, with Km values of 1.64, 1.87, 0.012, 0.014 mM, respectively. The ICHG showed a pronounced specificity for glucose in the 7-position of isoflavone and flavone conjugates and hydrolyzed effectively malonyl isoflavone glucosides as well as isoflavone glucosides with similar kinetics. Glucose and glucono-delta-lactone inhibited the enzyme competitively with Ki values of 84 mM and 23 mM, respectively. The enzyme did not require divalent cations for activity, and its activity was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, iodoacetic acid, and N-ethylmaleimide while reducing agents such as beta-mercaptoethanol, dithiothreitol, dithioerythritol, glutathione slightly activated the enzyme.
Yang
Liu
L
The State Key Laboratory for Biocontrol, Sun Yet-Sen University, Guangzhou, 510275, PR China.
Ning
Zhou S
ZS
Shi
Chen Z
CZ
Chang
Zhong Y
ZY
Huan
Liu Y
LY
D000645:Ammonium Sulfate; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007529:Isoflavones / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007529:Isoflavones; D001617:beta-Glucosidase; D005947:Glucose; D000645:Ammonium Sulfate