The glutamate decarboxylase (GAD) system of Lactobacillus brevis involves two isoforms of GAD, GadA and GadB, which catalyze the conversion of L-glutamate to γ-aminobutyric acid (GABA) in a proton-consuming reaction contributing to intracellular pH homeostasis. However, direct experimental evidence for detailed contributions of gad genes to acid tolerance and GABA production is lacking. Molecular analysis revealed that gadB is cotranscribed in tandem with upstream gadC, and that expression of gadCB is greatly upregulated in response to low ambient pH when cells enter the late exponential growth phase. In contrast, gadA is located away from the other gad genes, and its expression was consistently lower and not induced by mild acid treatment. Analysis of deletion mutations in the gad genes of L. brevis demonstrated a decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order of wild-type> ΔgadA> ΔgadB> ΔgadC> ΔgadAB, indicating that the GAD activity mainly endowed by GadB rather than GadA is an indispensable step in the GadCB mediated acid resistance of this organism. Moreover, engineered strains with higher GAD activities were constructed by overexpressing key GAD system genes. With the proposed two-stage pH and temperature control fed-batch fermentation strategy, GABA production by the engineered strain L. brevis 9530: pNZ8148-gadBC continuously increased reaching a high level of 104.38 ± 3.47 g/L at 72 h. This is the first report of the detailed contribution of gad genes to acid tolerance and GABA production in L. brevis. Enhanced production of GABA by engineered L. brevis was achieved, and the resulting GABA level was one of the highest among lactic acid bacterial species grown in batch or fed-batch culture.

Super-paramagnetic iron oxide nanoparticles (SPIONs)/gelatin (gel)/polyvinyl alcohol (PVA) nanoparticles were designed and synthesized by the co-precipitation method and further modified with gel and PVA. These nanoparticles were used for the removal of Cu(II) and Zn(II) from aqueous solutions. The adsorbents were rich in different functional groups for chemisorption and showed effective adsorption properties. The adsorption of Cu(II) and Zn(II) on the SPIONs/gel and SPIONs/gel/PVA materials were investigated with respect to pH, adsorption kinetics, and adsorption isotherms. The adsorption data was fitted to the Langmuir, Freundlich, and Sips models at the optimum pH 5.2 (±0.2) over 60 min; SPIONs/gel showed maximum adsorption capacities of 47.594 mg/g and 40.559 mg/g for Cu(II) and Zn(II); SPIONs/gel/PVA showed those of 56.051 mg/g and 40.865 mg/g, respectively. The experimental data fitted the pseudo-second-order model, indicating that the process followed chemical monolayer adsorption. In addition, the SPIONs/gel/PVA showed better stability and Cu(II) adsorption efficiency than SPIONs/gel.

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t_1/2) at 40 °C and a 5.3 °C increase in T₅₀¹⁰ compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (T_m) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

• A double promoter P_T7lac and P_BAD expression system was constructed for tunable control of GadB and GadC overexpression. • Broadening the active pH range of GadB and GadC towards neutral pH improving the GABA productivity. • A high efficient GABA synthesis system using engineered E. coli Lemo21(DE3) cells as the biocatalysts has been developed.