Preparation and characterization of glutamate decarboxylase from Enterococcus faecium
YANG Shengyuan1*, LIN Qian2, LIU Shumin1, SU Qiaoyun1, HUANG Huiling1
1(College of Food Science and Engineering, Lingnan Normal University, Zhanjiang 524048, China) 2(College of Biology and Pharmacy, Yulin Normal University, Yulin 537000, China)
Abstract: To develop high-quality glutamate decarboxylase (GAD), the affinity of cellulose-binding domain (CBD) and the self-cleavage of DnaB (an intein) were used for the construction of CBD-DnaB-GAD fusion enzyme using Enterococcus faecium as gadB donor, and the purification of GAD and its enzymatic properties were also investigated. The results showed that recombinant Escherichia coli GDMCC60446 carrying pRPOCDN-EfagadB could efficiently express CBD-DnaB-GAD, and the optimal solution for self-cleavage of DnaB was 0.2 mol/L phosphate buffer saline (pH 6.5, containing 0.5 mol/L NaCl and 1 mmol/L EDTA). The GAD, prepared by one-step purification and self-cleavage, showed a single band on SDS-PAGE electrophoresis with a molecular weight of 55.68 kDa, and consists of only one subunit. The purified GAD showed maximum activity at pH 5.0 and 55 ℃, and was stable at the range of pH 4.8 to 5.8 and -25 to 55 ℃. Its activity was hardly affected by 5 mmol/L of NaCl, CaCl2 or ethylene glycol, but was inactivated by 5 mmol/L of KCl, EDTA-2Na, ZnSO4, CuSO4, MnSO4, MgSO4, FeCl2, FeCl3, AlCl3, AgNO3 or Pb(CH3COO)2 at different levels. The purified GAD only catalyzed the decarboxylation of L-glutamic acid with apparent Km and Vmax at 10.51 mmol/L and 3.41 μmol/(mL·min) respectively, and the reaction activities were not inhibited by the substrate and product. The purity and enzymatic properties of GAD were excellent, and the preparation process was simple. Due to the high purity and excellent enzymatic properties of GAD, as well as the simple preparation process, this technology has potential to be applied to industrial production.
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2021, Vol. 47 
