Immobilization and enzymatic properties of glutamate decarboxylase from Enterococcus faecium by affinity adsorption on regenerated chitin

文献类型： 外文期刊

第一作者： Yang, Sheng-Yuan

作者： Yang, Sheng-Yuan;Liu, Shu-Min;Liang, Gui-Lian;Liu, Jiao-Fen;Liang, Zi-Zhan;Liang, Jia-Rong;Wu, Yan-Yan;Lin, Qian

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关键词： Immobilization; Enzymatic property; Chitin; Glutamate decarboxylase; Enterococcus faecium; Cellulose-binding domain; Gamma-aminobutyric acid

期刊名称：AMINO ACIDS （ 影响因子：3.52； 五年影响因子：3.6 ）

ISSN： 0939-4451

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收录情况： SCI

摘要： Glutamate decarboxylase (GAD, EC 4.1.1.15) is an important enzyme in gamma-aminobutyric acid biosynthesis and DL-glutamic acid resolution. In this study, the Enterococcus faecium-derived GAD was successfully immobilized by regenerated chitin (RC) via specific adsorption of cellulose-binding domain (CBD). The optimal binding buffer was 20 mmol/L phosphate buffer saline (pH 8.0), and the RC binding capacity was 1.77 +/- 0.11 mg(cbd-gad)/g(rc) under this condition. The ratio of wet RC and crude enzyme solution used for immobilization was recommended to 3:50 (g/mL). To evaluate the effect of RC immobilization on GAD, properties of the immobilize GAD (RC-CBD-GAD) were investigated. Results indicated RC-CBD-GAD was relatively stable at pH 4.4-5.6 and temperature - 20-40 degrees C, and the optimal reaction pH value and temperature were pH 4.8 and 50 degrees C, respectively. When it was reacted with 5 mmol/L of follow chemical reagents respectively, the activity of RC-CBD-GAD was hardly affected by EDTA, KCl, and NaCl, and significantly inactivated by AgNO3, MnSO4, MgSO4, CuSO4, ZnSO4, FeCl2, FeCl3, AlCl3, CaCl2, and Pb(CH3COO)(2). The apparent K-m and V-max were 28.35 mmol/L and 147.06 mu mol/(g(RC-CBD-GAD)center dot min), respectively. The optimum time for a batch of catalytic reaction without exogenous pH control was 2 h. Under this reaction time, RC-CBD-GAD had a good reusability with a half-life of 23 cycles, indicating that it was very attractive for GABA industry. As a novel, efficient, and green CBD binding carrier, RC provides an alternative way to protein immobilization.

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