Galactose Oxidase (GalOx) has gained significant interest in biocatalysis due to its ability for selective oxidation beyond the natural oxidation of galactose. However, the practical application of GalOx has been hindered by the limited availability of active and stable biocatalysts, as well as the inherent biochemical limitations such as oxygen (O2) dependency and the need for activation. In this study, we addressed these challenges by immobilizing GalOx into agarose-based and Purolite supports. Additionally, we identified and quantified the oxygen supply limitation into solid catalysts by intraparticle oxygen sensing showing a trade-off between the amount of protein loaded onto the solid support and the catalytic effectiveness. Furthermore, we coimmobilized a heme-containing protein along with the enzyme to function as an activator. To evaluate the application of the immobilized GalOx, we conducted the oxidation of galactose in an instrumented aerated reactor. The results showcased the efficient performance of the immobilized enzyme in the 8 h reaction cycle. Notably, the GalOx immobilized into dextran sulfate-activated agarose exhibited improved stability, overcoming the need for a soluble activator supply, and demonstrated exceptional performance in galactose oxidation. These findings offer promising prospects for the utilization of GalOx in technical biocatalytic applications.

The combination of polymers and low molecular weight (LMW) compounds is a powerful approach to prepare new supramolecular materials – i.e. hydrogels. Here we prepare two-component hydrogels made by a well-known and biologically-active polymer, hyaluronic acid (HA), and a dipeptide-based supramolecular gelator. We undertake a detailed study of different compositions including macroscopic (hydrogel formation, rheology) and micro/nanoscopic characterization (electron microscopy, X-ray powder diffraction). We observe that the two components mutually benefit in the new material: a minimum amout of HA (1-5 mol% of COOH groups) helps to reduce the polymorphism of the LMW component leading to reproducible hydrogels with improved mechanical properties; the LMW component network holds HA without the need of irreversible covalent crosslinking. These materials have a great potential for biomedical application as, for instance, extracellular matrix mimetics for cell growth. As a proof of concept, we have observed that this material is effective for cell growth in suspension and avoids cell sedimentation even in the presence of competing cell-adhesive surfaces, which can be of interest for advanced cell delivery techniques.

Most natural formate dehydrogenases (FDHs) exhibit NAD+ specificity, making it imperative to explore the engineering of FDH cofactor specificity for NADPH regeneration systems. The endogenous FDH of Komagataella phaffii (K. phaffii), termed KphFDH, is a typical NAD+-specific FDH. However, investigations into engineering the cofactor specificity of KphFDH have yet to be conducted. To develop an NADP+-specific variant of KphFDH, we selected D195, Y196, and Q197 as mutation sites and generated twenty site-directed variants. Through kinetic characterization, KphFDH/V19 (D195Q/Y196R/Q197H) was identified as the variant with the highest specificity towards NADP+, with a ratio of catalytic efficiency (kcat/KM)NADP+/(kcat/KM)NAD+ of 129.226. Studies of enzymatic properties revealed that the optimal temperature and pH for the reduction reaction of NADP+ catalyzed by KphFDH/V19 were 45°C and 7.5, respectively. The molecular dynamics (MD) simulation was performed to elucidate the mechanism of high catalytic activity of KphFDH/V19 towards NADP+. Finally, KphFDH/V19 was applied to an in vitro NADPH regeneration system with Meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH/H227V) . This study successfully created a KphFDH variant with high NADP+ specificity and demonstrated its practical applicability in an in vitro NADPH regeneration system.

α-Deuterated amino acids are valuable building blocks for developing deuterated drugs, and are important tools for studying biological systems. Biocatalytic deuteration represent an attractive strategy to directly access enantiopure α-deuterated amino acids. Here, we show that a PLP-dependent Mannich cyclase, LolT, involved in the biosynthesis of loline alkaloids, is capable of deuterating a diverse range of L-amino acids, including basic and acidic, nonpolar and polar, aliphatic and aromatic amino acids. Furthermore, complete deuteration of many amino acids can be achieved within minutes with exquisite control on the site- and stereoselectivity. During the course of this investigation, we also unexpectedly discovered that LolT exhibits β-elimination activity with L-cystine and O-acetyl-L-serine, confirming our previous hypothesis based on structural and phylogenetic analysis that LolT, a Cα-C bond forming enzyme, is evolved from a primordial Cβ-S lyase family. Overall, our study demonstrates that LolT is an extreme versatile biocatalyst, and can be used for not only heterocyclic quaternary amino acid biosynthesis, but also biocatalytic amino acid deuteration.

A detailed analysis of over 2000 biotechnology projects in India reveals the diversity of an emerging industry that spans not only human healthcare (medical devices, therapeutics, vaccines, regenerative medicine, …), but also (bio)agriculture (plant breeding and cloning, animal biotechnology, crop disease and pest control, …) and industrial biotechnology (fine chemicals, environmental, clean energy, …).

Abstract

A comprehensive analysis of 2165 projects funded by India's Department of Biotechnology since 2005 through private-public partnerships, and as of 2012 through the ‘Biotechnology Industry Research Assistance Council (BIRAC)’ until BIRAC's tenth anniversary at the end of March 2022 reveals details of the science and technology underpinning past and current biotechnology research and development projects in the country. They are led by human healthcare projects (74.9 % overall), of which medical technology (58.7 %) and therapeutics (24.5 %) are the main drivers, ahead of vaccines (4.3 %), regenerative medicine (3.9 %), public health (3.5 %) and others (5.1 %). Agricultural projects (15.2 % overall) have mainly been driven by plant breeding and cloning (24.6 %), animal biotechnology (20.4 %), agri-informatics (13.4 %), aquaculture (6.1 %), and (bio)fertilizers (4.3 %). The key components of industrial biotechnology (9.9 % overall) have been fine chemicals (44.7 %), environmental projects (23.3 %), clean energy (18.1 %) and industrial enzymes (12.1 %). Analysis of the projects funded pre- versus post-2017, compared to the distribution of equity funding as of early 2022 identifies trends in terms of growth areas and locations of industrial biotechnology projects and activities in India.

The conversion of novel olivetolic acid derivatives with the highly promiscuous prenyltransferase NphB is analyzed as a tool for the creation of synthetic cannabinoid libraries. By using in silico and in vitro experiments CBGA derivatives were synthesized and characterized as products of enzyme catalysis.

Abstract

NphB is an aromatic prenyltransferase with high promiscuity for phenolics including flavonoids, isoflavonoids, and plant polyketides. It has been demonstrated that cannabigerolic acid is successfully formed by the reaction catalysed by NphB using geranyl diphosphate and olivetolic acid as substrates. In this study, the substrate specificity of NphB was further determined by using olivetolic acid derivatives as potential substrates for the formation of new synthetic cannabinoids. The derivatives differ in the hydrocarbon chain attached to C6 of the core structure. We performed in silico experiments, including docking of olivetolic acid derivatives, to identify differences in their binding modes. Substrate acceptance was predicted. Based on these results, a library of olivetolic acid derivatives was constructed and synthesized by using different organic synthetic routes. Conversion was monitored in in vitro assays with purified NphB versions. For the substrates leading to a high conversion olivetolic acid-C8, olivetolic acid-C2 and 2-benzyl-4,6-dihydroxybenzoic acid, the products were further elucidated and identified as cannbigerolic acid derivatives. Therefore, these substrates show potential to be adapted in cannabinoid biosynthesis.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems

Cellobiose dehydrogenase serves as an auxiliary enzyme donating electrons to lytic polysaccharide monooxygenase in biomass depolymerization, as well as a biorecognition element in biosensors due to its electron transfer capability. The involvement of two amino acids in the interdomain electron transfer process is investigated in depth with stopped-flow spectrophotometry, small-angle X-ray scattering, multistate modeling, and molecular dynamics simulations.

Abstract

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway – its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

In this review, we survey mainly experimental but also theoretical work, focusing on radiation-induced intra- and inter-molecular transfer of charge, atoms, and energy within biomolecular systems in the gas phase. Building blocks of DNA, proteins, and saccharides, but also antibiotics are considered. The emergence of general processes as well as their timescales and mechanisms are highlighted.

Abstract

In biological tissues, ionizing radiation interacts with a variety of molecules and the consequences include cell killing and the modification of mechanical properties. Applications of biological radiation action are for instance radiotherapy, sterilization, or the tailoring of biomaterial properties. During the first femtoseconds to milliseconds after the initial radiation action, biomolecular systems typically respond by transfer of charge, atoms, or energy. In the condensed phase, it is usually very difficult to distinguish direct effects from indirect effects. A straightforward solution for this problem is the use of gas-phase techniques, for instance from the field of mass spectrometry. In this review, we survey mainly experimental but also theoretical work, focusing on radiation-induced intra- and inter-molecular transfer of charge, atoms, and energy within biomolecular systems in the gas phase. Building blocks of DNA, proteins, and saccharides, but also antibiotics are considered. The emergence of general processes as well as their timescales and mechanisms are highlighted.

The cRGD-functionalized nanoparticles obtained by chiral self-assembly with red emission and AIE characteristics could serve as efficient fluorescent probes for targeting cancer cells.

Abstract

We report a fluorescent dye TM by incorporating the tetraphenylethylene (TPE) and cholesterol components into perylene bisimides (PBI) derivative. Fluorescence emission spectrum shows that the dye has stable red emission and aggregation-induced emission (AIE) characteristics. The incorporation of cholesterol components triggers TM to show induced chirality through supramolecular self-assembly. The cRGD-functionalized nanoparticles were prepared by encapsulating fluorescent dyes with amphiphilic polymer matrix. The functionalized fluorescent organic nanoparticles exhibit excellent biocompatibility, large Stokes’ shift and good photostability, which make them effective fluorescent probes for targeting cancer cells with high fluorescence contrast.