Biosynthesis of γ-aminobutyric acid by a recombinant Bacillus subtilis strain expressing the glutamate decarboxylase gene derived from Streptococcus salivarius ssp thermophilus Y2

We expressed glutamate decarboxylase (GAD) from Streptococcus salivarius ssp. thermophilus Y2 in Bacillus subtilis to achieve synthesis of gamma-aminobutyric acid (GABA). Expression of the GAD gene in B. subtilis was achieved using the Escherichia coli T7 RNA polymerase (T7 RNAP) expression system. The culture reached maximal SY2-rGAD activity at 48 h of cultivation (16.2 U/mg protein), and after a four-step purification, the specific activity reached 163.4 U/mg protein. The molecular masses of SY2-rGAD under denaturing and native conditions were 53 kDa and 110 kDa, respectively, indicating that SY2-rGAD exists as a homodimer. Its optimum temperature and pH were 55 degrees C and 4.5, respectively. The purified SY2-rGAD was specific for L-glutamate (K-m = 2.72 mM, V-max = 165.8 mu mol/h/mg, K-cat = 5 S-1). The GABA-synthesizing ability of the recombinant non-growing B. subtilis is 512.9 mu mol/h/g (wet cells), which was more than 13-fold higher than that reported for S. thermophilus Y2 (39.2 mu mol/h/g). After 12 h, the GABA biosynthesized by this recombinant B. subtilis strain reached 5.26 g/L. To the best of our knowledge, the yield of GABA here is higher than that reported previously by B. subtilis. (C) 2014 Elsevier Ltd. All rights reserved.