Application of Berthelot Reaction in detection of glutamate decarboxylase from Lactococcus lactis SYFS1. 009

摘要

A colorimetric method with high sensitivity, reproducibility and low cost for determining glutamate decarboxylase (GAD) activity was developed based on the rationale of Berthelot reaction. The standard procedure for Berthelot reaction is given as follow; against ice-water bath, sample solution (0.3 ml) was mixed with 200 μL borate buffer (0.2 M, pH9.0) and 1000 μL 6% phenol solution, then 400 μL sodium hypochlorite was added followed by vigorous agitation. Color development was carried out in boiling water for 10 min followed by immediately cooling in ice-water bath for 20 min. The optical density is measured at 630 nm. For GAD assay, 200 μL substrate solution (0.2 M McDvaine buffer pH 4.7, containing 0.1 mM pyridoxal phosphate (PLP) and 10 mM L-monosodium glutamate (L-MSG)) was mixed with 1 ～100 μL enzyme preparation and incubated at 30°C. After enzymatic reaction, the optical densities were observed by standard procedure and interference by L-MSG was corrected. Gamma-aminobutyric acid (GABA) produced was calculated using the standard curve. Application of the assay method in GAD purification was also reported in this paper.