Search

γ-Aminobutyrate (GABA) is an important chemical in pharmaceutical field. The use of lactic acid bacteria as biocatalysts for the conversion of l -monosodium glutamate (MSG) into GABA opens interesting perspectives for the production of this functional compound. In this work, an engineered GABA high-producing strain Lactobacillus brevis GadA ΔC14 was constructed by overexpressing a C-terminally truncated GadA mutant, which is active in expanded pH range. After comparison with agar and κ-carrageenan, gellan gum was selected as the optimal immobilization support, and the properties of L. brevis GadA ΔC14 cells encapsulated in this hydrogel were examined. The optimum pH and temperature of immobilized cells were found to be 40°C and pH 4.4, respectively. It was also observed that operational and thermal stabilities of the cells were increased with immobilization. After ten consecutive reaction cycles, the total amounts of GABA produced by the immobilized cells summed up to 87.56 g/L under the optimum experimental conditions. Taken together, the improved stability and good usability make the immobilized L. brevis GadA ΔC14 cells more valuable for industrial applications. [ABSTRACT FROM AUTHOR]

• The GAD system in Lactococcus lactis subsp. lactis CV56 has been identified. • Recombinant GadB has been biochemically characterized. • Genetic engineering has been employed to increase the expression of GAD system genes in L. lactis NZ9000. • Enhanced production of GABA by using a two-stage pH control strategy. γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). Biosynthesis of GABA by lactic acid bacteria (LAB) has particularly attracted attention, as LAB are generally recognized as safe organisms. In this work, based on identification of biosynthetic gene cluster for GABA in Lactococcus lactis subsp. lactis CV56, the GadB has been heterologously expressed in Escherichia coli , purified and biochemically characterized. The recombinant enzyme existed as a homodimer under native conditions with a molecular mass of 115.03 kDa, and exhibited maximal activity at 50 °C and pH 4.7. The K m value of GadB was 3.9 mM for L-glutamate, with a limiting rate (V max) of 15.1 U/mg. Subsequently, the genetic engineering technique has been employed to increase the levels of expression of key GAD system genes from strain CV56 in the model LAB- L. lactis subsp. cremoris NZ9000. The fermentation results revealed that the coexpression of gadC and gadB made much more contribution to the overall yield of GABA than overexpression of any individual gene. Moreover, a novel two-stage pH control strategy for the selected strain L. lactis NZ9000/pNZ8148- gadBC was developed, and 25.61 g/L of GABA was finally achieved in the batch fermentation. [ABSTRACT FROM AUTHOR]