Expression and Transformation Characteristics of a Novel Glutamic Acid Decarboxylase LcGAD10s and Its Application on Sufu Processing.

Gamma-aminobutyric acid (GABA) is an important non-proteinogenic amino acid and a potent bioactive compound with many anti-hypertensive and anti-depressant activities. The bioconversion of GABA by glutamic acid decarboxylase (GAD) has been eagerly studied. Herein, novel pyridoxal-5-phosphate monohydrates (PLP)-dependent GAD, which is not quite similar to reporting, was cloned from and efficiently expressed in . The conveniently purified GAD (designated LcGAD10s) appeared as a single protein on SDS-PAGE with a molecular mass of 52.0 kDa. LcGAD10s exhibited a specific activity of 303.7 U/mg after purification by Ni-IDA affinity chromatography, with optimal activity at 55 °C and pH 5. LcGAD10s displayed excellent temperature (50 °C) and pH (4-8) stability which relative activity above 80% and 70%, respectively. The enzymatic activity was, respectively, increased and depressed by 130%, and 24% in the presence of Mn and Cu. Enzyme activity over 90% can be achieved by adding at least 25 mM of PLP. LcGAD10s was able to efficiently transform 15 g/L GABA with a single-factor optimized reaction of pH (5), temperature (50 °C), time (2 h), LcGAD10s dosage (0.4 U) and monosodium glutamate level (5 g/L). Additionally, LcGAD10s can be applied to a tofu fermentation system to achieve GABA conversion and achieved 14.9 mg/g of GABA conversion when added at 2 U/mL, which is higher than most of the commercial sufu and previous application reports, increasing its functional substances.