Properties of Glutamate Decarboxylase (gad) from Edible Mushroom

Enzymatic properties of glutamate decarboxylase (GAD) [EC4.1.1.15] obtained from the fruiting body of Flammulina velutipes were evaluated via protein isolation, purification, and characterization. To investigate the anatomical localization of GAD, crushed fruiting bodies were centrifuged; the supernatant and precipitate were subsequently subjected to enzyme reaction. GAD activity of the precipitate was much stronger than that of the supernatant. Although Nonidet P40 slightly solubilized GAD protein from cell wall fractions, most of the activity remained in the cell debris. A nearly homogeneous protein band was observed in SDSPAGE analysis after 10 treatments of solubilization and subsequent purification with ammonium sulfate precipitation and ultrafiltration. In cell wall-binding enzyme experiments, formation of amino butyric acid (GABA) was observed between pH 4 and 6, and the maximum GAD activity was observed at pH 6. However, GAD activity was lost after overnight dialysis against buffering of pH 6-10. The enzyme activity was optimum at 28°C and stable below 50°C. GAD of F. velutipes was specific for L-glutamate.