Facile Synthesis of Iron Carbide via Pyrolysis of Ferrous Fumarate for Catalytic CO2 Hydrogenation to Lower Olefins.

Hydrogenation of CO2 to olefin catalyzed by iron-based catalysts is a sustainable and important way to achieve carbon neutrality. In this study, iron-based catalysts were facilely prepared by direct pyrolysis of ferric fumarate (FF), which are applied to CO2 hydrogenation to olefin reaction to explore the effects of pyrolysis temperature and atmosphere on catalytic performance of the catalysts. Among them, NaFe-Air-400 catalyst exhibits the highest catalytic activity with 33.7 %, and light olefin selectivity reaches as high as 47.1 %. The catalytic performance of pyrolytic catalysts is better than that the impregnated NaFe catalyst on activated carbon (NaFe/AC). A series of XRD, Raman and SEM characterization results show a suitable pyrolysis temperature would promote the balance between amorphous carbon and graphene, which can affect the formation of FexCy phase, leading the distinctive activity and olefin selectivity. Hence, the presented one-step pyrolysis methodology would provide a facile and quick synthesis of highly-active iron-based catalyst design for CO2 conversion.

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.

Atomistic insight into the binding mode and self-regulation mechanism of IsPETase towards PET substrates with different polymerization degrees.

Poly(ethylene terephthalate) (PET) is one of the most widely used synthetic polyesters, however, its extensive use creates a long-term environmental burden. Unlike traditional recycling methods, biodegradation is a sustainable strategy. The emergence of PETase from Ideonella sakaiensis 201-F6 (IsPETase) has brought great potential for the industrialization of degradable PET. In this work, models of enzyme-substrate complexes with different degrees of polymerization were established to study the binding mode using molecular dynamics simulation. We found that the whole binding site can be further subdivided into three parts, including head, middle and tail binding regions. Most importantly, the presence of the middle region formed by both ends of Ser93 and Ser236 provides a potential possibility for the binding of substrates with different chain lengths, and exerts the self-regulation ability of enzymes to accommodate substrates. Meanwhile, the 'pocket bottom' Arg280 in the tail region echoes the 'pocket mouth' Trp185 in the head region, defining the substrate binding region. This work reveals the self-regulation of IsPETase, as well as the key residues for the substrate binding. The solution to these problems enables us to better understand the function of enzymes and design high-performance degradation enzymes, which is of great significance for industrial application research.

[Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability].

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

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.

Improved Stability and Catalytic Efficiency of ω-Transaminase in Aqueous Mixture of Deep Eutectic Solvents.

The efficient biosynthesis of chiral amines at an industrial scale to meet the high demand from industries that require chiral amines as precursors is challenging due to the poor stability and low catalytic efficiency of ω-transaminases (ω-TAs). Herein, this study adopted a green and efficient solvent engineering method to explore the effects of various aqueous solutions of deep eutectic solvents (DESs) as cosolvents on the catalytic efficiency and stability of ω-TA. Binary- and ternary-based DESs were used as cosolvents in enhancing the catalytic activity and stability of a ω-TA variant from Aspergillus terreus (E133A). The enzyme exhibited a higher catalytic activity in a ternary-based DES that was 2.4-fold higher than in conventional buffer. Moreover, the thermal stability was enhanced by a magnitude of 2.7, with an improvement in storage stability. Molecular docking studies illustrated that the most potent DES established strong hydrogen bond interactions with the enzyme's amino acid, which enhanced the catalytic efficiency and improved the stability of the ω-TA. Molecular docking is essential in designing DESs for a specific enzyme.

Turning thermostability of Aspergillus terreus (R)-selective transaminase At-ATA by synthetic shuffling.

As versatile and green biocatalysts for the asymmetric amination of ketones, the insufficient thermostability of transaminases always limits its broad application in the pharmaceutical and fine chemical industries. Here, synthetic shuffling technology was used to enhance stability of (R)-selective transaminase from Aspergillus terreus. The results showed that 30 out of 5000 mutants had improved thermostability by color-based screening method, among which mutants with residual enzyme activity higher than 50% at 45 °C for 10 min were selected for further analysis. Especially, the half-inactivation temperature (T5010), half-life (t1/2), and melting temperature (Tm) of the best mutant M14 (M280C-H210N-M150C-F115L) were 13.7 °C, 165.8 min, and 13.9 °C higher than that of the wild type (WT), respectively. M14 also exhibited a significant biocatalytic efficiency toward acetophenone and 1-acetylnaphthalene, the yield of which were 265.6% and 117.5% higher than WT, respectively. Based on molecular dynamics simulation, improved catalytic efficiency of M14 could be attributed to its increased hydrogen bonds interaction around the mutation sites. Additionally, the introduction of disulfide bond combined with above mutations has a synergistic effect on the improved protein thermostability.

QM/MM and MM MD simulations on decontamination of the V-type nerve agent VX by phosphotriesterase: toward a comprehensive understanding of steroselectivity and activity.

Due to deadly toxicity and high environmental stability of the nerve agent VX, an efficient decontamination approach is desperately needed in tackling its severe threat to human security. The enzymatic destruction of nerve agents has been generally considered as one of the most effective ways, and here the hydrolysis of VX by phosphotriesterase (PTE) was investigated by extensive QM/MM and MM MD simulations. The hydrolytic cleavage of P-S by PTE is a two-step process with the free energy spans of 15.8 and 26.0 kcal mol-1 for the RP- and SP-enantiomer VX, respectively, and such remarkable stereospecificity of VX enantiomers in the enzymatic degradation is attributed to their conformational compatibility with the active pocket. The structurally less adaptive SP-enantiomer allows one additional water molecule to enter the binuclear zinc center and remarkably facilitates the release of the degraded product. Overall, the rate-limiting steps in the enzymatic degradation of VX by PTE involve the degraded product release of the RP-enantiomer and the enzymatic P-S cleavage of the SP-enantiomer. Further computational analysis on the mutation of selected residues also revealed that H257Y, H257D, H254Q-H257F, and L7ep-3a variants allow more water molecules to enter the active site, which improves the catalytic efficiency of PTE, as observed experimentally. The present work provides mechanistic insights into the stereoselective hydrolysis of VX by PTE and the activity manipulation through the active-site accessibility of water molecules, which can be used for the enzyme engineering to defeat chemical warfare agents.

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.

Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM-1s-1, respectively, with the specific activity of 26.67 µM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBΔC11C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.

Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

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.

Exploring the contributions of two glutamate decarboxylase isozymes in Lactobacillus brevis to acid resistance and γ-aminobutyric acid production.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Contribution of the activated catalase to oxidative stress resistance and γ-aminobutyric acid production in Lactobacillus brevis.

Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound which can paradoxically produce themselves and lead to the growth arrest and cell death. To counteract the potentially toxic effects of this compound, the gene katE encoding a heme-dependent catalase (CAT) belonging to the family of monofunctional CATs was cloned from Lactobacillus brevis CGMCC1306. The enhanced homologous CAT expression was achieved using the NICE system. L. brevis cells with overexpressed CAT showed 685-fold and 823-fold higher survival when exposed to 30mmol/L of H2O2 and long-term aerated stress (after 72h), respectively, than that of the wild type cells. Furtherly, the effects of activated CAT on GABA production in L. brevis were investigated. A GABA production level of 66.4g/L was achieved using two-step biotransformation that successively employed the growing and resting cells derived from engineering L. brevis CAT. These results demonstrated clearly that overexpression of the KatE gene in L. brevis led to a marked increased survival in oxidizing environment, and shed light on a novel feasible approach to enhance the GABA production level by improving the antioxidative properties.