Escherichia coli glutamate decarboxylase (GAD) converts glutamate into gamma-aminobutyric acid (GABA) through decarboxylation using proton as a co-substrate. Since GAD is active only at acidic conditions even though pH increases as the reaction proceeds, the conventional practice of using this enzyme involved the use of relatively high concentration of buffers, which might complicate the downstream purification steps. Here we show by simulation and experiments that the free acid substrate, glutamic acid, rather than its monosodium salt can act as a substrate and buffer at the same time. This yielded the buffer- and salt-free synthesis of GABA conveniently in a batch mode. Furthermore, we engineered GAD to hyper active ones by extending or reducing the length of the enzyme by just one residue at its C-terminus. Through the buffer-free reaction with engineered GAD, we could synthesize 1 M GABA in 3 h, which can be translated into a space-time yield of 34.3 g/L/h. (C) 2013 Elsevier Inc. All rights reserved.