Immobilization of β-Galactosidase by Encapsulation of Enzyme-Conjugated Polymer Nanoparticles Inside Hydrogel Microparticles

Increasing the shelf life of enzymes and making them reusable is a prominent topic in biotechnology. The encapsulation inside hydrogel microparticles (HMPs) can enhance the enzyme’s stability by preserving its native conformation and facilitating continuous biocatalytic processes and enzyme recovery. In this study, we present a method to immobilize β-galactosidase by, first, conjugating the enzyme onto the surface of polymer nanoparticles, and then encapsulating these enzyme-conjugated nanoparticles (ENPs) inside HMPs using microfluidic device paired with UV-LEDs. Polymer nanoparticles act as anchors for enzyme molecules, potentially preventing their leaching through the hydrogel network especially during swelling. The affinity binding (through streptavidin-biotin interaction) was used as an immobilization technique of β-galactosidase on the surface of polymer nanoparticles. The hydrogel microparticles of roughly 400 μm in size (swollen state) containing unbound enzyme and ENPs were produced. The effects of encapsulation and storage in different conditions were evaluated. It was discovered that the encapsulation in acrylamide (AcAm) microparticles caused an almost complete loss of enzymatic activity. Encapsulation in poly(ethylene glycol) (PEG)-diacrylate microparticles, on the other hand, showed a residual activity of 15–25%, presumably due to a protective effect of PEG during polymerization. One of the major factors that affected the enzyme activity was presence of photoinitiator exposed to UV-irradiation. Storage studies were carried out at room temperature, in the fridge and in the freezer throughout 1, 7 and 28 days. The polymer nanoparticles showcased excellent immobilization properties and preserved the activity of the conjugated enzyme at room temperature (115% residual activity after 28 days), while a slight decrease was observed for the unbound enzyme (94% after 28 days). Similar trends were observed for encapsulated ENPs and unbound enzyme. Nevertheless, storage at −26°C resulted in an almost complete loss of enzymatic activity for all samples.

DEVELOPMENT AND RESEARCH OF POLYMER COMPOSITE MATERIAL WITH CEFAZOLIN BASED ON POLYURETHANE WITH ISOCYANURATE FRAGMENTS

Institute of Chemistry of Macromolecular Chemistry NAS of Ukraine, 48, Kharkivske shose, Kyiv, 02160, Ukraine Developed polymer composite material with Cefazolin based on polyurethane with isocyanurate fragments (PU), synthesized on the basis of polyoxypropylene glycol (POPG 1000), TDI (2,4;2,6-toluene diisocyanate) and 2,4,6-triisocyanate(trishexamethylene) isocyanate (ICC, Tolonate ™ HDT-LV, MM 1200) at a ratio of NCO: OH = 1: 1 in the medium N,N’-dimethylacetamide (DMAA) and Cefazolin (CFZ). CFZ is immobilized on isocyanurate-containing polyurethane by introducing a solution of CFZ with DMA into the reaction the amount of 5 wt. %. The synthesized polymeric material (PU-CFZ) represents a transparent film of yellow color with physical and mechanical characteristics: σ = 0,15 MPa; ε = 63.40%. According to IR spectroscopy, Cefazolin is immobilized on the polymer matrix by physically due to hydrogen interactions. The influence of biological medium 199 (BM 199) on the structure and properties of PU-CFZ during incubation for 1, 3 and 6 months was studied. It was found that after incubation in BM 199, the structure of PU-CFZ changes as a result of the interaction of enzyme molecules that are part of BM 199 with urethane and amide groups of PU. According to the results of physical and mechanical studies, after incubation in BM 199 for 6 months, PU-CFZ samples are characterized by tensile strength of 0.36 MPa and elongation at break of 98%, ie retain sufficient performance for use in biological objects for up to 6 months. Composite materials with Cefazolin are able to prolong the release of the drug for 28 days in an amount of about 30%, which is sufficient to provide a local therapeutic effect. According to the results of the cytotoxicity study of the developed material by in vitro tissue culture, it was found that the composite material based on isocyanurate-containing polyurethane with Cefazolin is biocompatible.

Comparison of Spatial Structures and Packaging of Phosphorybosil Pyrophosphate Synthetase 2 from Thermus thermophilus HB27 in Rhombohedral and Tetragonal Crystals

We report the spatial structure of phosphoribosyl pyrophosphate synthetase 2 from the thermophilic bacterium Thermus thermophilus HB27 (TthPRPPS2) obtained at a 1.85 Å resolution using a diffraction set collected from rhombohedral crystals (space group R32-h), grown with lithium sulfate as a precipitant. This crystal structure was compared with the structure of TthPRPPS2, previously obtained at a 2.2 Å resolution using diffraction sets from the tetragonal crystals (space group P41212), grown with ammonium sulfate as a precipitant. The comparison of these structures allows the study of the differences between protein molecules in both crystalline structures, as well as the packaging of enzyme molecules in crystals of both spatial groups. Our results may contribute to the research of the structural basis of catalytic activity and substrate specificity of this enzyme.

Magnetic Nanoparticle-Containing Supports as Carriers of Immobilized Enzymes: Key Factors Influencing the Biocatalyst Performance

In this short review (Perspective), we identify key features of the performance of biocatalysts developed by the immobilization of enzymes on the supports containing magnetic nanoparticles (NPs), analyzing the scientific literature for the last five years. A clear advantage of magnetic supports is their easy separation due to the magnetic attraction between magnetic NPs and an external magnetic field, facilitating the biocatalyst reuse. This allows for savings of materials and energy in the biocatalytic process. Commonly, magnetic NPs are isolated from enzymes either by polymers, silica, or some other protective layer. However, in those cases when iron oxide NPs are in close proximity to the enzyme, the biocatalyst may display a fascinating behavior, allowing for synergy of the performance due to the enzyme-like properties shown in iron oxides. Another important parameter which is discussed in this review is the magnetic support porosity, especially in hierarchical porous supports. In the case of comparatively large pores, which can freely accommodate enzyme molecules without jeopardizing their conformation, the enzyme surface ordering may create an optimal crowding on the support, enhancing the biocatalytic performance. Other factors such as surface-modifying agents or special enzyme reactor designs can be also influential in the performance of magnetic NP based immobilized enzymes.

Non-Heating Alternating Magnetic Field Nanomechanical Stimulation of Biomolecule Structures via Magnetic Nanoparticles as the Basis for Future Low-Toxic Biomedical Applications

The review discusses the theoretical, experimental and toxicological aspects of the prospective biomedical application of functionalized magnetic nanoparticles (MNPs) activated by a low frequency non-heating alternating magnetic field (AMF). In this approach, known as nano-magnetomechanical activation (NMMA), the MNPs are used as mediators that localize and apply force to such target biomolecular structures as enzyme molecules, transport vesicles, cell organelles, etc., without significant heating. It is shown that NMMA can become a biophysical platform for a family of therapy methods including the addressed delivery and controlled release of therapeutic agents from transport nanomodules, as well as selective molecular nanoscale localized drugless nanomechanical impacts. It is characterized by low system biochemical and electromagnetic toxicity. A technique of 3D scanning of the NMMA region with the size of several mm to several cm over object internals has been described.

The effect of antioxidants on xanthine oxidase activity in fresh ovine milk

In the present, the consequences of nitrate pollution of the environment are very pronounced. In humans and animals, microorganisms can reduce nitrates to nitrites, which cause cancer. Purified and homogeneous xanthine oxidase (XO) of cow's milk can restore these compounds, which makes the article extremely relevant. The purpose of the article is to determine the effect of antioxidants on the activity of xanthine oxidase in fresh ovine milk. Various natural and artificial antioxidants were examined for the detection of xanthine oxidase (XO) activity in ovine milk. Among the natural antioxidants, L-cysteine was more effective in the stabilization of XO in heated milk. XO of sheep milk activated by heat treatment in the presence of cysteine and molybdenum became able to convert nitrate and nitrite to nitric oxide (NO). Therefore, L-cysteine was used for double purposes: as the protector of enzyme active center against the oxidation during heat treatment of milk and as a reagent for S-nitrosothiol formation. Hypoxanthine, as a natural substrate of XO, is an effective electron donor for nitrate reductase (NR) and nitrite reductase (NiR) activities. Heat treatment of the milk in the presence of exogenous lecithin increased the activity of NR and NiR of XO and CysNO formation. Thus, during the heat treatment: a) excess of exogenous phospholipids disintegrates the structure of milk fat globule membrane (MFGM) and b) enzyme molecules denatured partially and their active center became available for exogenous cysteine, molybdenum, hypoxanthine, and nitrate or nitrite.

Analytical Parameters of a Novel Glucose Biosensor Based on Grafted PFM as a Covalent Immobilization Technique

Bioanalytical methods, in particular electrochemical biosensors, are increasingly used in different industrial sectors due to their simplicity, low cost, and fast response. However, to be able to reliably use this type of device, it is necessary to undertake in-depth evaluation of their fundamental analytical parameters. In this work, analytical parameters of an amperometric biosensor based on covalent immobilization of glucose oxidase (GOx) were evaluated. GOx was immobilized using plasma-grafted pentafluorophenyl methacrylate (pgPFM) as an anchor onto a tailored HEMA-co-EGDA hydrogel that coats a titanium dioxide nanotubes array (TiO2NTAs). Finally, chitosan was used to protect the enzyme molecules. The biosensor offered outstanding analytical parameters: repeatability (RSD = 1.7%), reproducibility (RSD = 1.3%), accuracy (deviation = 4.8%), and robustness (RSD = 2.4%). In addition, the Ti/TiO2NTAs/ppHEMA-co-EGDA/pgPFM/GOx/Chitosan biosensor showed good long-term stability; after 20 days, it retained 89% of its initial sensitivity. Finally, glucose concentrations of different food samples were measured and compared using an official standard method (HPLC). Deviation was lower than 10% in all measured samples. Therefore, the developed biosensor can be considered to be a reliable analytical tool for quantification measurements.

Selective degradation of synthetic polymers through enzymes immobilized on nanocarriers

In order to develop new sustainable and reusable concepts for the degradation of omnipresent industrial plastics, immobilization of (bio)catalysts on nanocarriers offers unique opportunities for selective depolymerization and catalyst recovery. In this study, enzymes (lipase and cutinase) were covalently immobilized on carrier nanoparticles (SiO2 and Fe3O4@SiO2) through 3-(aminopropyl)trimethoxysilane and glutaraldehyde linkers forming a stable bond to enzyme molecules. The presence of enzymes on the surface was confirmed by zeta potential and XPS measurements, while their degradation activity and long-term stability of up to 144 h was demonstrated by the conversion of 4-nitrophenyl acetate to 4-nitrophenol. Furthermore, enzymatic decomposition (hydrolysis/oxidation) of electrospun polycaprolactone fiber mats was verified through morphological (SEM) and weight loss studies, which evidently showed a change in the fiber morphology due to enzymatic degradation and accordingly a weight loss. Graphic abstract

Biochemical Background in Mitochondria Affects 2HG Production by IDH2 and ADHFE1 in Breast Carcinoma

Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, we provide a background for vital mitochondrial production of 2HG in breast cancer cells with outcomes towards cancer biology and possible future diagnosis of breast carcinoma.