Specific stereoisomer is paramount as it is vital for optimizing drug efficacy and safety. The quest for the isolation of desired stereoisomer of active pharmaceutical ingredients or key intermediates drives innovation in drug synthetic and biocatalytic methods. Chiral phosphoramidate is an important building block for the synthesis of antiviral drugs such as remdesivir and sofosbuvir. Given the clinical potency of the (Sp)-diastereomer of the drugs, an enzyme capable of completely hydrolyzing the (Rp)-diastereomer is needed to achieve the purified diastereomers via biocatalytic reaction. In this study, protein engineering of phosphotriesterase (PTE) was aimed to improve the specificity. Employing rational design and site-directed mutagenesis, we generated a small library comprising 24 variants for activity screening. Notably, W131M and I106A/W131M variants demonstrated successful preparation of pure (Sp)-diastereomer of remdesivir and sofosbuvir precursors within a remarkably short hydrolysis time (<20 min). Our work unveils a promising methodology for producing pure stereoisomeric compounds, utilizing novel biocatalysts to enable the chemoenzymatic synthesis of phosphoramidate nucleoside prodrugs.

Perillyl alcohol (POH) is a secondary metabolite of plants. POH and its derivatives are known to be effective as an anticancer treatment. In this study, oxidative derivatives of POH, which are difficult to synthesize chemically, were synthesized using the engineered bacterial cytochrome P450 BM3 (CYP102A1) as a biocatalyst. The activity of wild-type (WT) CYP102A1 and 29 engineered enzymes toward POH was screened using a high-performance liquid chromatography. They produced one major product. Among them, the engineered CYP102A1 M601 mutant with seven mutations (R47L/F81I/F87V/E143G/L150F/L188Q/E267V) showed the highest conversion, 6.4-fold higher than the WT. Structure modeling using AlphFold2 and PyMoL suggests that mutations near the water channel may be responsible for the increased catalytic activity of the M601 mutant. The major product was identified as a POH-8,9-epoxide by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis. The optimal temperature and pH for the product formation were 35 °C and pH 7.4, respectively. The kcat and Km of M601 were 540 min-1 and 2.77 mM, respectively. To improve POH-8,9-epoxide production, substrate concentration and reaction time were optimized. The optimal condition for POH-8,9-epoxide production by M601 was 5.0 mM POH, pH 7.4, 35 ℃, and 6 h reaction, which produced the highest concentration of 1.72 mM. Therefore, the biosynthesis of POH-8,9-epoxide using M601 as a biocatalyst is suggested to be an efficient and sustainable synthetic process that can be applied to chemical and pharmaceutical industries.

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg-1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency (kcat/Km) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v/v ethanol and 70% v/v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

Phosphorus (P) is essential for biological systems, playing a pivotal role in energy metabolism and forming crucial structural components of DNA and RNA. Yet its bioavailable forms are scarce. Phytate, a major form of stored phosphorus in cereals and soils, is poorly bioavailable due to its complex structure. Phytases, enzymes that hydrolyze phytate to release useable phosphorus, are vital in overcoming this limitation and have significant biotechnological applications. This study employed novel method to isolate and characterize bacterial strains capable of metabolizing phytate as the sole carbon and phosphorus source from the Andes mountains soils. Ten strains from the genera Klebsiella and Chryseobacterium were isolated, with Chryseobacterium sp. CP-77 and Klebsiella pneumoniae CP-84 showing specific activities of 3.5 ± 0.4 nkat/mg and 40.8 ± 5 nkat/mg, respectively. Genomic sequencing revealed significant genetic diversity, suggesting CP-77 may represent a novel Chryseobacterium species. A fosmid library screening identified several phytase genes, including a 3-phytase in CP-77 and a glucose 1-phosphatase and 3-phytase in CP-84. Phylogenetic analysis confirmed the novelty of these enzymes. These findings highlight the potential of phytase-producing bacteria in sustainable agriculture by enhancing phosphorus bioavailability, reducing reliance on synthetic fertilizers, and contributing to environmental management. This study expands our biotechnological toolkit for microbial phosphorus management and underscores the importance of exploring poorly characterized environments for novel microbial functions. The integration of direct cultivation with metagenomic screening offers robust approaches for discovering microbial biocatalysts, promoting sustainable agricultural practices, and advancing environmental conservation.

The most common heterocyclic aromatic molecule with potential uses in industry and medicine is quinoline. Its chemical formula is C9H7N, and it has a distinctive double-ring structure with a pyridine moiety fused with a benzene ring. Various synthetic approaches synthesize quinoline derivatives. These approaches include solvent-free synthetic approach, mechanochemistry, ultrasonic, photolytic synthetic approach, and microwave and catalytic synthetic approaches. One of the important synthetic approaches is a catalyst-based synthetic approach in which different catalysts are used such as silver-based catalysts, titanium-based nanoparticle catalysts, new iridium catalysts, barium-based catalysts, iron-based catalysts, gold-based catalysts, nickel-based catalyst, some metal-based photocatalyst, α-amylase biocatalyst, by using multifunctional metal-organic framework-metal nanoparticle tandem catalyst etc. In the present study, we summarized different catalyst-promoted reactions that have been reported for the synthesis of quinoline. Hopefully, the study will be helpful for the researchers.

This study reports the genome sequence data of a novel Stenotrophomonas sepilia Alg010 strain isolated from Sargassum seaweed waste accumulated on the coastline of Barbados. The genome sequence data was obtained via sequencing of the genomic DNA of this isolate with Illumina NextSeq2000 platform and paired-end library preparation protocol. The resulting reads were assembled with the SPAdes Genome Assembler (ver 3.15.4) and annotated with the DDBJ Fast Annotation and Submission Tool. The genome size of this novel isolate was recorded as 4,515,447 bp with a coverage of 270×, a GC content of 66.6 % and a gap ratio of 0.027 %. The lengths of the longest and the N50 contigs were estimated as 246,749 bp and 81,982 bp, respectively. The genome contains 2 rRNA, 66 tRNA, 2 CRISPR, 86 contigs and 4024 CDSs (coding sequences) with a coding ratio of 88.9 %. The annotation of the CDSs for COG (cluster of orthologous groups) and for subsystem features indicated that the metabolism and the amino acids and derivatives were the most dominant categories, respectively. The annotation of the genome via dbCAN3 server for carbohydrate-active genes revealed 98 genes encoding the six functional classes of carbohydrate-active enzymes. The genome sequence data is available in NCBI GenBank with the accession number BTRJ00000000.

The biosynthetic investigations of microbial natural products continuously provide powerful biocatalysts for the preparation of valuable chemicals. Practical methods for preparing (S)-3-aminopiperidine-2,6-dione (2), the pharmacophore of thalidomide (1) and its analog drugs, are highly desired. To develop a biocatalyst for producing (S)-2, we dissected the domain functions of IdgS, which is responsible for the biosynthesis of indigoidine (3), a microbial blue pigment that consists of two 2-like moieties. Our data supported that the L-glutamine tethered to the indigoidine assembly line is first offloaded and cyclized by the thioesterase domain to form (S)-2, which is then dehydrogenated by the oxidation (Ox) domain and finally dimerized to yield 3. Based on this, we developed an IdgS-derived enzyme biocatalyst, IdgS-Ox* R539A, for preparing enantiomerically pure (S)-2. As a proof of concept, one-pot chemoenzymatic synthesis of 1 was achieved by combining the biocatalytic and chemical approaches.

In this study, a biodegradable and eco-friendly biocatalyst (eggshell/Fe3O4) was synthesized utilizing eggshell impregnated with Fe3O4 nanoparticles. The characterization of prepared catalyst was carried out by Fourier transform infrared radiation (FT-IR), scanning electron microscopy (SEM), X-ray Diffraction (XRD), energy-dispersive X-ray (EDX), thermal gravimetric analysis-differential thermogravimetry (TGA-DTG), vibrating sample magnometer (VSM), and atomic force microscopy (AFM). The eggshell/Fe3O4 biocatalyst was served in multi-component reactions (MCRs) for the synthesis of 2-amino thiophene derivatives from variety aromatic aldehydes, malononitrile, ethyl acetoacetate, and sulfur (S8). To achieve optimal reaction conditions, a thorough examination was conducted on key factors, such as the solvent type, reaction time and temperature, and the ratio of eggshell to Fe3O4. The findings suggest that high yield product can be obtained at microwave temperature (MW) in EtOH solvent within 10 min. Additionally, the eggshell/Fe3O4 biocatalyst exhibited high catalytic activity, which was sustained over the five cycles, without any significant decline in its performance.

Here, we developed magnetically recoverable biocatalysts based on magnetite nanoparticles coated with an ultra-thin layer (about 0.9 nm) of chitosan (CS) ionically cross-linked by sodium tripolyphosphate (TPP). Excessive CS amounts were removed by multiple washings combined with magnetic separation. Glucose oxidase (GOx) was attached to the magnetic support via the interaction with N-hydroxysuccinimide (NHS) in the presence of carbodiimide (EDC) leading to a covalent amide bond. These steps result in the formation of the biocatalyst for D-glucose oxidation to D-gluconic acid to be used in the preparation of pharmaceuticals due to the benign character of the biocatalyst components. To choose the catalyst with the best catalytic performance, the amounts of CS, TPP, NHS, EDC, and GOx were varied. The optimal biocatalyst allowed for 100% relative catalytic activity. The immobilization of GOx and the magnetic character of the support prevents GOx and biocatalyst loss and allows for repeated use.

This study unveils an innovative method for synthesizing coumarin S-glycosides, employing original biocatalysts able to graft diverse carbohydrate structures onto 7-mercapto-4-methyl-coumarin in one-pot reactions. The fluorescence properties of the generated thio-derivatives were assessed, providing valuable insights into their potential applications in biological imaging or sensing. In addition, the synthesized compounds exhibited no cytotoxicity across various human cell lines. This research presents a promising avenue for the development of coumarin S-glycosides, paving the way for their application in diverse biomedical research areas.