Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.).
BACKGROUND
γ-Aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro.


RESULTS
The molecular mass of the enzyme estimated by Sephadex G-100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg(2+), Cu(2+), Fe(3+), aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA-Na(2)), L-cysteine and β-mercaptoethanol. The K(m) value of DAO was 0.23 mmol L(-1) for putrescine and 0.96 mmol L(-1) for spermidine. However, the enzyme did not degrade spermine.


CONCLUSIONS
DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine.
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
Runqiang
R
Yang
Hui
H
Chen
Yongbin
Y
Han
Zhenxin
Z
Gu
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018525:Germination / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011700:Putrescine / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*; D013095:Spermidine / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D031307:Vicia faba / Q000201:enzymology*; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis*
D004791:Enzyme Inhibitors; D005680:gamma-Aminobutyric Acid; D006631:Amine Oxidase (Copper-Containing); D013095:Spermidine; D011700:Putrescine