Stereoselective synthesis of (R)-(+)-1-(1-naphthyl)ethylamine by ω-amine transaminase immobilized on amino modified multi-walled carbon nanotubes and biocatalyst recycling.

Immobilized enzymes exhibit favorable advantages in biocatalysis, such as high operation stability, feasible reusability, and improved organic solvents tolerance. Herein, an immobilized ω-amine transaminase AtATA@MWCNTs-NH2 is successfully prepared using amino modified multi-walled carbon nanotubes as carrier and glutaraldehyde as crosslinker. Under the optimum immobilization conditions, the activity recovery is 78.7%. Compared with purified enzyme AtATA, AtATA@MWCNTs-NH2 possesses superior stability, even in harsh conditions (e.g., high temperature, acidic or alkali environment, and different kind of organic solvents). To simplify the separation and extraction of products, we choose methanol (10%, v/v) as the cosolvent, replacing DMSO (20%, v/v) in our previous work, for the catalytic reaction of AtATA@MWCNTs-NH2. AtATA@MWCNTs-NH2 can be used for stereoselective synthesis (R)-(+)- 1(1-naphthyl)ethylamine ((R)-NEA) for 15 cycles, with the e.e.p (enantiomeric excess) > 99.5%. The catalytic process of AtATA@MWCNTs-NH2 achieves cycle production of (R)-NEA using methanol as cosolvent.

Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine.

BACKGROUND: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential. MAIN METHODS AND RESULTS: The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (kcat /Km ) toward 1-acetylnaphthalene and the stability (half-life t1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value >99.5%) within 10 h under 25% DMSO. CONCLUSION: The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

Food-grade γ-aminobutyric acid production by immobilized glutamate decarboxylase from Lactobacillus plantarum in rice vinegar and monosodium glutamate system.

OBJECTIVES: γ-amino butyric acid (GABA) is a non-protein amino acid, considered a potent bioactive compound. This study focused on biosynthesis of food-grade GABA by immobilized glutamate decarboxylase (GAD) from Lactobacillus plantarum in the rice vinegar and monosodium glutamate (MSG) reaction system. RESULTS: The gene encoding glutamate decarboxylase (GadB) from L. plantarum has been heterologously expressed in Lactococcus lactis and biochemically characterized. Recombinant GadB existed as a homodimer, and displayed maximal activity at 40 °C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of GadB for L-Glu was 22.33 mM and 62.4 mM-1 min-1, respectively, with a specific activity of 24.97 U/mg protein. Then, purified GadB was encapsulated in gellan gum beads. Compared to the free enzyme, immobilized GadB showed higher operational and storage stability. Finally, 9.82 to 21.48 g/L of GABA have been acquired by regulating the amounts of catalyst microspheres ranging from 0.5 to 0.8 g (wet weight) in 0.8 mL of the designed rice vinegar and MSG reaction system. CONCLUSIONS: The method of production GABA by immobilized GadB microspheres mixed in the rice vinegar and MSG reaction system is introduced herein for the first time. Especially, the results obtained here meet the increased interest in the harnessing of biocatalyst to synthesize food-grade GABA.

Biosynthesis of γ-aminobutyrate by engineered Lactobacillus brevis cells immobilized in gellan gum gel beads.

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 GadAΔC14 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 GadAΔC14 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 GadAΔC14 cells more valuable for industrial applications.

Physiology-Oriented Engineering Strategy to Improve Gamma-Aminobutyrate Production in Lactobacillus brevis.

Gamma-aminobutyrate (GABA) is an important chemical in the pharmaceutical field. GABA-producing lactic acid bacteria (LAB) offer the opportunity of developing this health-oriented product. In this study, the gadA, gadB, gadC, gadCB, and gadCA gene segments of Lactobacillus brevis were cloned into pMG36e, and strain Lb. brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA production after analysis of GAD activities. Subsequently, a physiology-oriented engineering strategy was adopted to construct an FoF1-ATPase deficient strain NRA6 with higher GAD activity. As expected, strain NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42% GABA conversion rate in GYP fermentation medium, which is 1.22-fold higher than that obtained by the wild-type strain in the same condition. This work demonstrates how the acid stress response mechanisms of LAB can be employed to develop cell factories with improved production efficiency and contributes to research into the development of the physiology-oriented engineering.