Screening Of Glutamic Acid Decarboxylase Producing Fungi And Enzyme Purification

Glutamic acid decarboxylase (GAD) is a key enzyme, which only catalyze L-glutamic acid decarboxylase generated gamma-aminobutyric acid (GAB A). GAB A has widely apply potential, such as health care, food, chemical industry, agriculture and so on. For that, there is a great significance to research GAB A. In this paper,71microorganisms were isolated directly from cooked Pu’er tea, six Fort tea and brick tea. By color method screening, colorimetric method re-screening, one strain producing GAD relatively high was screened, which is strain4. In order to further determine that strain4producing glutamic acid decarboxylase.2,4-2nitro fluorobenzene derivative method is presented in this paper, the conditions of ultra high performance liquid chromatography for determining glutamate decarboxylase (GAD) activity were optimized. Based on the morphology and fungal ITS analysis, the strain4was preliminary identified as a species of Phomopsis. As a starting strain4, the response surface method about fermentation medium and conditions for optimization was used. The GAD produced by strain4in liquid fermentation was isolated and purified by a three-step:ammonium sulfate precipitation, DEAE-sephrose Fast Flow aion-exchange chromatography, Sephadex G-150gel penetration chromatography. After the three-step purification, GAD was showed a single band on SDS-PAGE electrophoresis, the molecular mass of the GAD was about44.3kDa., its purity and yield were6.57folds and39.88%of the crude extract respectively. The enzyme properties of GAD were stduied, its optimal temperature and pH of the purified respectively was40℃and5.5.