Search Results (58)

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

Three fungal strains were employed for the stereoselective oxidation of the cheap and commercially available substrate 2-phenylethanol, which resulted in chiral building blocks being received. The whole-cell biocatalysts were as follows: Beauveria bassiana DSM 1344, Beauveria brongniartii DSM 6651, and Rhizopus arrhizus DSM 1185. The main product of Beauveria bassiana bioconversion was 1-phenylethane-1,2-diol, obtained, depending on the form of the biocatalyst, as an R-enantiomer (e.g., 99.9%) with fresh biomass application or as a racemic mixture in cases of immobilization in agar-agar. The best and most innovative results for the synthesis of the R-enantiomer of diol were received under precisely defined conditions as a result of a scaling study conducted on an automatic batch reactor. This is a pioneering result, since, in previous studies, fresh mycelium of Aspergillus niger resulted in this product being received as the (S) enantiomer. Also, the use of Rhizopus arrhizus DSM 1185 (immobilized in polyurethane foams) presented important results, as the bioconversion of phenyl ethanol led, indeed, to the racemic mixture of 1-phenylethane-1,2-diol but was accompanied by a noticeable tyrosol synthesis, which had not been reported previously. Full article

Several chemical and biocatalytic methods have been described for chiral γ-lactams syntheses. However, only one biocatalytic method has been reported for γ⁴-lactam resolution, while γ²- and γ³-lactams have not been reported. On the other hand, its resolution through biocatalysts is complicated since enzymes such as ENZA-1 (Rhodococcus equi NCIB 40213) and ENZA-20 (Pseudomonas solanacearum NCIB 40249) are difficult to obtain. Therefore, in this paper, the resolution of γ-lactams 7-9 was carried out through a hydrolysis reaction using the commercially available enzyme CaLB. Full article

This study aimed to optimize the kinetic resolution of building blocks for the synthesis of β-blockers using Candida rugosa lipases, which could be potentially used to synthesize enantiomerically pure β-blockers further. Reaction mixtures were incubated in a thermostated shaker. Qualitative and quantitative analyses of the reaction mixtures were performed using chiral stationary phases and the UPLC-IT-TOF system. Of the 24 catalytic systems prepared, a system containing lipase from Candida rugosa MY, [EMIM][BF₄] and toluene as a two-phase reaction medium and isopropenyl acetate as an acetylating agent was optimal. This resulted in a product with high enantiomeric purity produced via biotransformation, whose enantioselectivity was E = 67.5. Using lipases from Candida rugosa enables the enantioselective biotransformation of the β-blockers building block. The biocatalyst used, the reaction environment, and the acetylating agent significantly influence the efficiency of performer kinetic resolutions. The studies made it possible to select an optimum system, a prerequisite for obtaining a product of high enantiomeric purity. As a result of the performed biotransformation, the (S)-enantiomer of the β-blocker derivative was obtained, which can be used to further synthesize enantiomerically pure β-blockers. Full article

This study explored the enantiocomplementary bioreduction of substituted 1-(arylsulfanyl)propan-2-ones in batch mode using four wild-type yeast strains and two different recombinant alcohol dehydrogenases from Lactobacillus kefir and Rhodococcus aetherivorans. The selected yeast strains and recombinant alcohol dehydrogenases as whole-cell biocatalysts resulted in the corresponding 1-(arylsulfanyl)propan-2-ols with moderate to excellent conversions (60–99%) and high selectivities (ee > 95%). The best bioreductions—in terms of conversion (>90%) and enantiomeric excess (>99% ee)—at preparative scale resulted in the expected chiral alcohols with similar conversion and selectivity to the screening reactions. Full article

Flavin-containing monooxygenase from Methylophaga sp. (mFMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored mFMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis (NiFMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of mFMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations. Full article

Endophytic microorganisms are promising sources for new biocatalysts as they must deal with their host plants’ chemicals by developing adaptative strategies, such as enzymatic pathways. As part of our efforts in selecting endophytic strains as biocatalysts, this study describes the screening of endophytic fungi isolated from Handroanthus impetiginosus leaves for selective bioreduction of Acetophenone. The bioreductions were monitored by chiral gas chromatography and conducted to the selection of the endophyte Talaromyces sp. H4 as capable of reducing acetophenone to (S)-1-phenylethanol in excellent conversion and enantiomeric excess rates. The influence of seven parameters on the stereoselective bioreduction of acetophenone by Talaromyces sp. H4 was studied: reaction time, inoculum charge, shaking, pH, temperature, substrate concentration, and co-solvent. The optimal conditions were then used to reduce substituted acetophenones and Acetophenone scale-up, which furnished (S)-1-Phenylethanol in 73% yield and 96% ee. The results highlight the endophytic fungus Talaromyces sp. H4 as an excellent biocatalyst for stereoselective reduction of prochiral carbonyls. Full article

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed. Full article

A self-sufficient bifunctional enzyme integrating reductive amination and coenzyme regeneration activities was developed and successfully employed to synthesize (S)-cyclopropylglycine with an improved reaction rate 2.1-fold over the native enzymes and a short bioconversion period of 6 h at a high substrate concentration of 120 g·L⁻¹ and space–time yield of (S)-cyclopropylglycine up to 377.3 g·L⁻¹·d⁻¹, higher than that of any previously reported data. Additionally, (S)-cyclopropylglycine could be continuously synthesized for 90 h with the enzymes packed in a dialysis tube, providing 634.6 g of (S)-cyclopropylglycine with >99.5% ee and over 95% conversion yield up to 12 changes. These results confirmed that the newly developed NADH-driven biocatalytic system could be utilized as a self-sufficient biocatalyst for industrial application in the synthesis of (S)-cyclopropylglycine, which provides a chiral center and cyclopropyl fragment for the frequent synthesis of preclinical/clinical drug molecules. Full article

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base—pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (ee_p = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity. Full article

In lipase-catalyzed kinetic resolutions (KRs), the choice of immobilization support and acylating agents (AAs) is crucial. Lipase B from Candida antarctica immobilized onto magnetic nanoparticles (CaLB-MNPs) has been successfully used for diverse KRs of racemic compounds, but there is a lack of studies of the utilization of this potent biocatalyst in the KR of chiral amines, important pharmaceutical building blocks. Therefore, in this work, several racemic amines (heptane-2-amine, 1-methoxypropan-2-amine, 1-phenylethan-1-amine, and 4-phenylbutan-2-amine, (±)-1a–d, respectively) were studied in batch and continuous-flow mode utilizing different AAs, such as diisopropyl malonate 2A, isopropyl 2-cyanoacetate 2B, and isopropyl 2-ethoxyacetate 2C. The reactions performed with CaLB-MNPs were compared with Novozym 435 (N435) and the results in the literature. CaLB-MNPs were less active than N435, leading to lower conversion, but demonstrated a higher enantiomer selectivity, proving to be a good alternative to the commercial form. Compound 2C resulted in the best balance between conversion and enantiomer selectivity among the acylating agents. CaLB-MNPs proved to be efficient in the KR of chiral amines, having comparable or superior properties to other CaLB forms utilizing porous matrices for immobilization. An additional advantage of using CaLB-MNPs is that the purification and reuse processes are facilitated via magnetic retention/separation. In the continuous-flow mode, the usability and operational stability of CaLB-MNPs were reaffirmed, corroborating with previous studies, and the results overall improve our understanding of this potent biocatalyst and the convenient U-shape reactor used. Full article

Clopidogrel is a chiral compound widely used as an antiplatelet medication that lowers the risk of blood clots, strokes, and heart attacks. The main aim of the study presented herein was to obtain (S)-clopidogrel, which is commercially available in treatments, via the kinetic resolution of racemic clopidogrel carboxylic acid with the use of lipase from Candida rugosa and a two-phase reaction medium containing an ionic liquid. For this purpose, the enantioselective biotransformation of clopidogrel carboxylic acid and chiral chromatographic separation with the use of a UPLC-MS/MS system were optimized. The best kinetic resolution parameters were obtained by using a catalytic system containing lipase from Candida rugosa OF as a biocatalyst, cyclohexane and [EMIM][BF4] as a two-phase reaction medium, and methanol as an acyl acceptor. The enantiomeric excess of the product was ee_p = 94.21% ± 1.07 and the conversion was c = 49.60% ± 0.57%, whereas the enantioselectivity was E = 113.40 ± 1.29. The performed study proved the possibility of obtaining (S)-clopidogrel with the use of lipase as a biocatalyst and a two-phase reaction medium containing an ionic liquid, which is in parallel with green chemistry methodology and does not require environmentally harmful conditions. Full article

Epoxide hydrolases are attractive and industrially important biocatalysts. They can catalyze the enantioselective hydrolysis of epoxides to the corresponding diols as chiral building blocks for bioactive compounds and drugs. In this review article, we discuss the state of the art and development potential of epoxide hydrolases as biocatalysts based on the most recent approaches and techniques. The review covers new approaches to discover epoxide hydrolases using genome mining and enzyme metagenomics, as well as improving enzyme activity, enantioselectivity, enantioconvergence, and thermostability by directed evolution and a rational design. Further improvements in operational and storage stabilization, reusability, pH stabilization, and thermal stabilization by immobilization techniques are discussed in this study. New possibilities for expanding the synthetic capabilities of epoxide hydrolases by their involvement in non-natural enzyme cascade reactions are described. Full article

Aminotransferases (ATs) are pyridoxal 5′-phosphate-dependent enzymes that catalyze the reversible transfer of an amino group from an amino donor to a keto substrate. ATs are promising biocatalysts that are replacing traditional chemical routes for the production of chiral amines. In this study, an in silico-screening of a metagenomic library isolated from the Curonian Lagoon identified 11 full-length fold type IV aminotransferases that were successfully expressed and used for substrate profiling. Three of them (AT-872, AT-1132, and AT-4421) were active toward (R)-methylbenzylamine. Purified proteins showed activity with L- and D-amino acids and various aromatic compounds such as (R)-1-aminotetraline. AT-872 and AT-1132 exhibited thermostability and retained about 55% and 80% of their activities, respectively, even after 24 h of incubation at 50 °C. Active site modeling revealed that AT-872 and AT-4421 have an unusual active site environment similar to the AT of Haliscomenobacter hydrossis, while AT-1132 appeared to be structurally related to the AT from thermophilic archaea Geoglobus acetivorans. Thus, we have identified and characterized PLP fold type IV ATs that were active toward both amino acids and a variety of (R)-amines. Full article

The biocatalytic asymmetric reduction of prochiral ketones for the production of enantiopure alcohols is highly desirable due to its inherent advantages over chemical methods. In this study, a new bacterial strain capable of transforming ketones to corresponding alcohols with high activity and excellent enantioselectivity was discovered in a soil sample. The strain was subsequently identified as Bacillus cereus TQ-2 based on its physiological characteristics and 16S rDNA sequence analysis. Under optimized reaction conditions, the resting cells of B. cereus TQ-2 converted acetophenone to enantioenriched (R)-1-phenylethanol with 99% enantiometric excess following anti-Prelog’s rule, which is scarce in biocatalytic ketone reduction. The optimum temperature for the cells was 30 °C, and considerable catalytic activity was observed over a broad pH range from 5.0 to 9.0. The cells showed enhanced catalytic activity in the presence of 15% (v/v) glycerol as a co-substrate. The catalytic activity can also be substantially improved by adding Ca²⁺ or K⁺ ions. Moreover, the B. cereus TQ-2 cell was highly active in reducing several structurally diverse ketones and aldehydes to form corresponding alcohols with good to excellent conversion. Our study provides a versatile whole-cell biocatalyst that can be used in the asymmetric reduction of ketones for the production of chiral alcohol, thereby expanding the biocatalytic toolbox for potential practical applications. Full article