Search Results (39)

Chiral amines are vital structural motifs in pharmaceuticals and agrochemicals, where enantiomeric purity governs bioactivity and environmental behavior. We identified a novel (R)-selective amine transaminase (MwoAT) from Mycobacterium sp. via genome mining, which exhibits activity toward the synthesis of the chiral amine (R)-1-methyl-3-phenylpropylamine. The enzyme displayed optimal activity at pH 7.0 and 40 °C, with high thermostability and solvent tolerance. Using an AlphaFold3-guided semi-rational engineering strategy integrating molecular docking, alanine scanning, and saturation mutagenesis, residue L175 was pinpointed as critical for substrate binding. The resulting L175G variant exhibited a 2.1-fold increase in catalytic efficiency (k_cat/K_m) and improved thermal stability. Applied to the asymmetric synthesis of (R)-1-methyl-3-phenylpropylamine—a precursor for the antihypertensive drug dilevalol and potential scaffold for crop protection agents—the mutant achieved 26.4% conversion with ≥99.9% ee. The enzyme also accepted several ketones relevant to agrochemical synthesis, underscoring its versatility. This work delivers an engineered biocatalyst for sustainable chiral amine production and demonstrates an AI-assisted protein engineering framework applicable to both medicinal and agricultural chemistry. Full article

Deep eutectic solvents (DESs) have emerged as prominent, environmentally benign substitutes for traditional solvents and catalysts in organic synthesis, notably in the synthesis of amines, pivotal structures in many industrial sectors. Their distinctive physicochemical attributes—including negligible volatility, exceptional thermal stability, and adjustable polarity—render them particularly advantageous for facilitating a broad spectrum of amination reactions. DESs can serve dually as reaction media and as intrinsic catalytic systems, accelerating reaction kinetics without necessitating supplementary catalysts or severe reaction conditions. They are especially efficacious in processes such as reductive amination, transamination, and multicomponent transformations, often affording superior yields and streamlining product isolation. The extensive hydrogen-bonding network intrinsic to DESs is believed to mediate crucial mechanistic steps, frequently obviating the requirement for external additives. Moreover, DESs are recyclable and exhibit compatibility with a diverse array of substrates, encompassing bio-derived and pharmaceutical intermediates. Full article

The enzyme ω-transaminase (ω-TA) has garnered significant attention due to its capacity to catalyze the synthesis of chiral amines with high efficiency. Nevertheless, the lack of stability of ω-TA and the difficulty of recycling and reuse are still challenges that limit its application. This study developed a novel magnetic nanocellulose composite carrier (NNC@Fe₃O₄@Ni), synthesized from microcrystalline cellulose via low-eutectic solvent treatment, amine modification, and metal hybridization. The NNC@Fe₃O₄@Ni was characterized by FTIR, XPS, XRD, BET, and VSM. Additionally, the performance and catalytic behavior of the immobilized enzyme were investigated. The results revealed that NNC@Fe₃O₄@Ni exhibited a high specific surface area, superparamagnetism, and dual-site functionality (amine/Ni²⁺). Response Surface Methodology (RSM) optimized the carrier-enzyme interaction parameters, yielding optimal immobilization conditions: a mass ratio of 50.8 mg g⁻¹, temperature of 12.5 °C, and duration of 58.6 min, achieving 82.91% enzyme activity recovery. Compared to free enzymes, the immobilized variant demonstrated enhanced catalytic stability, with expanded optimal pH (9.0) and temperature (30 °C). Thermal stability assessments showed 84.39% activity retention after 5 h at 30 °C and 90.30% residual activity post-120 h storage. The catalyst maintained >80% efficiency over 10 reuse cycles. These findings confirm the efficacy of magnetic nanocellulose carriers in enhancing ω-TA stability, reusability, and catalytic performance, offering a viable strategy for industrial biocatalytic processes. Full article

ω-Transaminases are biocatalysts capable of asymmetrically synthesizing high-value chiral amines through the reductive amination of carbonyl compounds, and they are ubiquitously distributed across diverse microorganisms. Despite their broad natural occurrence, the industrial utility of naturally occurring ω-transaminases remains constrained by their limited catalytic efficiency toward sterically bulky substrates. Over recent decades, the use of structure-guided molecular modifications, leveraging three-dimensional structures, catalytic mechanisms, and machine learning-driven predictions, has emerged as a transformative strategy to address this limitation. Notably, these advancements have unlocked unprecedented progress in the asymmetric synthesis of bulky chiral amines, which is exemplified by the industrial-scale production of sitagliptin using engineered ω-transaminases. This review systematically explores the structural and mechanistic foundations of ω-transaminase engineering. We first delineate the substrate binding regions of these enzymes, focusing on their defining features such as substrate tunnels and dual pockets. These structural elements serve as critical targets for rational design to enhance substrate promiscuity. Next, we dissect the catalytic and substrate recognition mechanisms of (S)- and (R)-ω-transaminases. Drawing on these insights, we consolidate recent advances in engineering ω-transaminases to highlight their performance in synthesizing bulky chiral amines and aim to guide future research and the industrial implementation of tailored ω-transaminases. Full article

Sitagliptin, an important anti-diabetic drug, can be obtained using transaminase (TA) enzymes, which are known to be promising biocatalysts for the production of highly enantiopure amines under mild reaction conditions. In an industrial context, the use of immobilized enzymes can provide several advantages, such as the improved stability of the biocatalyst and easy product recovery. In this study, a new commercially available transaminase enzyme to produce sitagliptin was immobilized on inorganic and organic supports using two different approaches: adsorption and covalent bond formation. Among the inorganic media, non-functionalized silica gel was chosen for its stability and competitive cost. A range of commercially available resins with different functionalities have also been selected for their characteristics that can meet industrial standards. The immobilized biocatalysts were first tested in the transamination of acetophenone as a model substrate, which obtains, in most cases, higher conversions with respect to soluble enzymes. The best results in the enantioselective synthesis of sitagliptin were achieved with the sample immobilized on the epoxy- and octadecyl-functionalized methacrylic resin, which allowed the complete conversion of the corresponding ketone and high enantioselectivity (>99% ee). Moreover, the recycling of the supported enzyme could be performed in a continuous flow system without loss of activity for five consecutive runs. Full article

Amine transaminases (ATA) are critical players in producing non-canonical amino acids, essential building blocks in pharmaceuticals and fine chemicals. Significant progress has been made in discovering and engineering enzymes in this field, enhancing their use in organic synthesis. However, challenges such as co-factor regeneration, substrate, and product inhibition remain significant limitations to widespread industrial enzyme application. (Chemo-)enzymatic cascades offer efficient and environmentally friendly pathways for synthesizing amino acids, reducing the need for multiple synthesis steps and saving the purification of intermediates. This review focuses specifically on the synthesis of non-canonical amino acids, emphasizing the use of enzymatic and chemoenzymatic cascades involving ATA. Full article

Albizia odoratissima is a deciduous tree species belonging to the family Leguminosae. It is widely distributed in the southern subtropical and tropical areas of China and has important ecological and economic value. The growth and metabolic processes of A. odoratissima are affected by drought stress, but the molecular mechanisms remain unknown. Therefore, this study investigated the physicochemical properties, gene expression, and metabolites of A. odoratissima seedlings under drought stress. The results show that, in leaves of A. odoratissima seedlings, drought stress reduced the moisture content, chlorophyll content, photosynthetic efficiency, superoxide dismutase (SOD) activity, and gibberellin (GA) and indoleacetic acid (IAA) contents while increasing the catalase (CAT) and peroxidase (POD) activities and malondialdehyde (MDA), proline, soluble sugar, and soluble protein contents. Within the CK5 (Day 5 of control group) vs. T5 (Day 5 of drought treatment), CK10 vs. T10, CK15 vs. T15, and CK20 vs. T20 groups (CK: control group; T: drought treatment), a total of 676 differentially expressed genes (DEGs) were upregulated and 518 DEGs were downregulated, and a total of 228 and 143 differential accumulation metabolites (DAMs) were identified in the CK10 vs. T10 and CK20 vs. T20 groups. These were mainly involved in the amino acid and alkaloid metabolism pathways in the leaves of the A. odoratissima seedlings. In the amino acid and alkaloid biosynthesis pathways, the relative expression levels of the AoproA (Aod04G002740, ORTHODONTIC APPLIANCE), AoOAT (Aod07G015970, ORNITHINE-OXO-ACID TRANSAMINASE), and AoAOC3 (Aod12G005010/08G003360/05G023920/08G003000/08G003010, AMINE OXIDASE COPPER CONTAINING 3) genes increased, which concurrently promoted the accumulation of arginine, proline, piperine, cadaverine, and lysine. Furthermore, some key transcription factors in the response to drought were identified in the leaves using the weighted gene co-expression network analyses (WGCNA) method. These findings reveal that A. odoratissima seedlings respond to drought stress by improving the capacities of the antioxidant system and secondary metabolism. Full article

The combination of wet-lab experimental data on multi-site combinatorial mutations and machine learning is an innovative method in protein engineering. In this study, we used an innovative sequence-activity relationship (innov’SAR) methodology based on novel descriptors and digital signal processing (DSP) to construct a predictive model. In this paper, 21 experimental (R)-selective amine transaminases from Aspergillus terreus (AT-ATA) were used as an input to predict higher thermostability mutants than those predicted using the existing data. We successfully improved the coefficient of determination (R²) of the model from 0.66 to 0.92. In addition, root-mean-squared deviation (RMSD), root-mean-squared fluctuation (RMSF), solvent accessible surface area (SASA), hydrogen bonds, and the radius of gyration were estimated based on molecular dynamics simulations, and the differences between the predicted mutants and the wild-type (WT) were analyzed. The successful application of the innov’SAR algorithm in improving the thermostability of AT-ATA may help in directed evolutionary screening and open up new avenues for protein engineering. Full article

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5′-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure–function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine—a reversible inhibitor and analogue of (R)-1-phenylethylamine—a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the β-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds. Full article

Building blocks with amine functionality are crucial in the chemical industry. Biocatalytic syntheses and chemicals derived from renewable resources are increasingly desired to achieve sustainable production of these amines. As a result, renewable materials such as furfurals, especially furfurylamines like 5-(hydroxymethyl)furfurylamine (HMFA) and 2,5-di(aminomethyl)furan (DAF), are gaining increasing attention. In this study, we identified four different amine transaminases (ATAs) that catalyze the reductive amination of 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF). We successfully immobilized these ATAs on glutaraldehyde-functionalized amine beads using multiple binding and on amine beads by site-selective binding of the unique Cα-formylglycine within an aldehyde tag. All immobilized ATAs were efficiently reused in five repetitive cycles of reductive amination of HMF with alanine as co-substrate, while the ATA from Silicibacter pomeroyi (ATA-Spo) also exhibited high stability for reuse when isopropylamine was used as an amine donor. Additionally, immobilized ATA-Spo yielded high conversion in the batch syntheses of HMFA and DAF using alanine (87% and 87%, respectively) or isopropylamine (99% and 98%, respectively) as amine donors. We further demonstrated that ATA-Spo was effective for the reductive amination of HMF with alanine or isopropylamine in continuous-flow catalysis with high conversion up to 12 days (48% and 41%, respectively). Full article

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. Full article

Pyridoxal-5′-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from Bacillus subtilis. However, at least two groups of D-amino acid transaminases differing in the active site organization are known today. Here, we present a detailed study of D-amino acid transaminase from the gram-negative bacterium Aminobacterium colombiense with a substrate binding mode different from that for the transaminase from B. subtilis. We study the enzyme using kinetic analysis, molecular modeling, and structural analysis of holoenzyme and its complex with D-glutamate. We compare the multipoint binding of D-glutamate with the binding of other substrates, D-aspartate and D-ornithine. QM/MM MD simulation reveals that the substrate can act as a base and its proton can be transferred from the amino group to the α-carboxylate group. This process occurs simultaneously with the nucleophilic attack of the PLP carbon atom by the nitrogen atom of the substrate forming gem-diamine at the transimination step. This explains the absence of the catalytic activity toward (R)-amines that lack an α-carboxylate group. The obtained results clarify another substrate binding mode in D-amino acid transaminases and underpinned the substrate activation mechanism. Full article

Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HA_GA-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from Luminiphilus syltensis. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChem^TM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability. Full article

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme. Full article

D-amino acids are valuable building blocks for the synthesis of biologically active compounds and pharmaceuticals. The asymmetric synthesis of chiral amino acids from prochiral ketones using stereoselective enzymes is a well-known but far from exhausted approach for large-scale production. Herein, we investigated a pyridoxal-5′-phosphate-dependent D-amino acid transaminase from Haliscomenobacter hydrossis as a potential biocatalyst for the enzymatic asymmetric synthesis of optically pure aliphatic and aromatic D-amino acids. We studied the catalytic efficiency and stereoselectivity of transaminase from H. hydrossis in the amination of aliphatic and aromatic α-keto acids, using D-glutamate as a source of the amino group. We constructed a one-pot three-enzyme system, which included transaminase and two auxiliary enzymes, hydroxyglutarate dehydrogenase, and glucose dehydrogenase, to produce D-amino acids with a product yield of 95–99% and an enantiomeric excess of more than 99%. We estimated the stability of the transaminase and the cofactor leakage under reaction conditions. It was found that a high concentration of α-keto acids as well as a low reaction temperature (30 °C) can reduce the cofactor leakage under reaction conditions. The obtained results demonstrated the efficiency of transaminase from H. hydrossis in the asymmetric synthesis of enantiomerically pure D-amino acids. Full article