Biosynthesis of γ-aminobutyric acid by a recombinant Bacillus subtilis strain expressing the glutamate decarboxylase gene derived from Streptococcus salivarius ssp. thermophilus Y2
2014
Zhang, Chong | Lü, Jing | Chen, Lin | Lu, Fengxia | Lu, Zhaoxin
We expressed glutamate decarboxylase (GAD) from Streptococcus salivarius ssp. thermophilus Y2 in Bacillus subtilis to achieve synthesis of γ-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 48h of cultivation (16.2U/mg protein), and after a four-step purification, the specific activity reached 163.4U/mg protein. The molecular masses of SY2-rGAD under denaturing and native conditions were 53kDa and 110kDa, respectively, indicating that SY2-rGAD exists as a homodimer. Its optimum temperature and pH were 55°C and 4.5, respectively. The purified SY2-rGAD was specific for L-glutamate (Km=2.72mM, Vmax=165.8μmol/h/mg, Kcat=5S−1). The GABA-synthesizing ability of the recombinant non-growing B. subtilis is 512.9μmol/h/g (wet cells), which was more than 13-fold higher than that reported for S. thermophilus Y2 (39.2μmol/h/g). After 12h, the GABA biosynthesized by this recombinant B. subtilis strain reached 5.26g/L. To the best of our knowledge, the yield of GABA here is higher than that reported previously by B. subtilis.
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