Proteins play a major role in biosensors in which they provide catalytic activity and specificity in molecular recognition. The immobilization process is however far from straightforward as it often affects the protein functionality. An extensive interaction of the protein with the surface or a significant surface crowding can lead to changes in the mobility and conformation of the protein structure. This review will provide an insight of how the analysis of the physico-chemical features of the protein surface features before the immobilization process can help to identify the optimal immobilization approach to preserve the functionality of the protein when on the surface of the biosensor.

Phosphatidylserine (PS) has significant applications in various sectors, such as the medical and food industries. However, its production relies heavily on phospholipase D (PLD), a crucial tool that is hindered by issues like poor stability and irrecoverability. Immobilization presents itself as an effective solution to overcome these limitations. In this study, magnetic graphene oxide (MGO) modified with amino (-NH₂) were synthesized and utilized for PLD immobilization. The activity of the immobilized PLD (MGO-PLD) reached 3062 U/g_MGO, with a specific activity of 33.9 U/mg_PLD, virtually identical to that of the free PLD. MGO-PLD was utilized to synthesize PS efficiently in a biphasic system. Under optimal conditions, PS yield reached 18.66 g/L, with a conversion rate of 92.8% and a productivity of 3.11 g/L/h. Notably, MGO-PLD retained an impressive PS conversion rate of 77.4% even after seven repetitive usages. Moreover, MGO-PLD displayed enhanced thermal and pH resistance properties compared to free PLD, alongside an augmented storage stability. Post a 8-week preservation at 4°C, its residual activity was able to maintain at 76.3%. This study provides a sustainable and highly efficient pathway for biocatalytic synthesis of PS.

Removal of nitrogen compound in wastewater has been successfully developed with various activated sludge based processes. The microorganism immobilized on media enhanced biological efficiency by increase in biomass concentration, however the microbial community composition in the media was little revealed. A modified Ludzack-Ettinger (MLE) process was operated with immobilized media with polyvinyl alcohol and polyethylene glycol. MLSS in aerobic reactor was maintained at 50,000 mg/L and 40,000 mg/L in anoxic reactor by the media. 99% of ammonium in influent was oxidized but organic nitrogen resulted from microbial growth reduced total oxidation rate during the operation. Nitrate reduction rate increased by the addition of glucose for C/N ratio adjustment to 4.5. Based on total nitrogen concentration, the removal efficiency of nitrogen was 48.2% after C/N ratio adjustment, which showed the mid-range of nitrogen removal efficiency in MLE. Microbial community composition was compared between aerobic and anoxic media by NGS technique with V3-V4 region of 16S rRNA gene. Proteobacteria was the dominant phylum both in aerobic and anoxic media, and the ratio was 39.7% in aerobic media and 65.9% in anoxic media. Bacteroidetes was secondly largest phylum. The dominant genera in aerobic media were Nitrospira and Povalibacter. Ratio of nitrification-related genera was 45%. On the contrary, the dominant genera in anoxic media were Desulfomicrobium, Desulfobulbus, and Methyloversatilis in sequence of dominance. Total genera related with denitrification, including Dechloromonas and Flavobacterium amounted 63%. Population of microorganisms in each reactor was compared in terms of diversity by QIIME 2 algorithm. The Chao1 index values of α-diversity were 606.05 and 415.53 for aerobic and anoxic media, respectively, which showed higher population diversity in aerobic media than in anoxic. The microbiological community on the immobilized aerobic and anoxic media would help future microbial studies in wastewater treatment process.

The usability of glass fibers as immobilization support with porous open structure has been explored. We have developed a method for immobilization of β-galactosidase on special glass fiber rolls. The new method provides a simple, non-expensive and industrially applicable method. Glutaraldehyde was used as a non-specific crosslinking agent for covalent binding of β-galactosidase on modified glass fibers. The efficiency of immobilization was tested with the known hydrolysis of lactose. All experiments were performed in a continuous laboratory reactor. The effect of reaction temperature (20, 25, and 30) °C, substrate flow rate (1, 2, and 3) mL/min, and pH of the reaction medium (6, 7, and 8) on the conversion was studied. The reaction efficiency was monitored by measuring the glucose concentration with a spectrophotometer. High immobilization efficiency and enzyme activity and stability were obtained. The optimum reaction temperature, substrate flow rate and pH were found. The activity and stability of the enzyme entrapped on the glass fiber rolls remained almost unchanged during reuse, which is a good prospect for potential industrial applications.

Food production heavily depends on ammonia-containing fertilizers to improve crop yield and profitability. However, ammonia production is challenged by huge energy demand, the release of ~2% of global CO2, and political instability. To mitigate this challenge, many research efforts have developed bioprocessing technologies to make biological ammonia. This review presents three different biological approaches that drive the biochemical mechanisms to convert nitrogen gas, bioresources, or waste to biological ammonia. The use of advanced technologies— enzyme immobilization and microbial bioengineering – enhanced bio-ammonia production. This review also highlighted some challenges and research gaps that require researchers’ attention for bio-ammonia to be industrially pragmatic.

A recent breakthrough in preparation of immobilized enzyme based biocatalysts achieving highly enhanced enzymatic activities and stabilities has become a great alternative to conventional immobilization techniques. The functional hybrid nanobiocatalysts (FHNs) fabricated in this immobilization composed of organic components (amino acid, peptide, protein, enzyme and plant extract) and inorganic components (various metal ions) give flower-like morphology with narrow size distribution and porous structure. The enzyme incorporated FHNs exhibite greatly enhanced catalytic activities and stabilities compared to free and conventionally immobilized enzymes under various experimental conditions. In addition to that, the FHNs consisting of other organic components act as Fenton-like reagents and show peroxidase-like activity owing to presence of metal ions and porous structure in the FHNs. This report basically focuses on preparation, characterization, and bioanalytical applications of the FHNs and explain mechanism of the FHNs formation and thier enhanced activities and stabilities.

Enzymes serve as biocatalysts for innumerable important reactions; however, their application has limitations, which could be overcome by using appropriate immobilization strategies. Here, a new support for immobilizing enzymes is proposed. This hybrid organic-inorganic support is composed of chitosan—a natural, nontoxic, biodegradable, and edible biopolymer—and sodium polyphosphate, which was the inorganic component. Lipase B from Candida antarctica (CALB) was immobilized in microspheres by encapsulation using these polymers. The characterization of the composites (by infrared spectroscopy, thermogravimetric analysis, and confocal Raman microscopy) confirmed the hybrid nature of the support, whose external part consisted of polyphosphate and core was composed of chitosan. The immobilized enzyme had the following advantages: possibility of enzyme reuse, easy biocatalyst recovery, increased resistance to variations in temperature (activity declined from 60°C and the enzyme was inactivated at 80°C), and increased catalytic activity in the transesterification reactions. The encapsulated enzymes were utilized as biocatalysts for transesterification reactions to produce the compound responsible for the aroma of jasmine.

This study reports rapid hydrolysis, acylation, and lipolysis with lipase B from Candida antarctica (CalB) and nanofructosome encapsulated CalB (CalB@NF) enzymes immobilized on silica-coated magnetic nanoparticles (Si-MNPs). Enzymatic hydrolysis was confirmed for conversion to p-nitrophenol (p-NP, λex=400 nm) from p-nitrophenyl butyrate (p-NPB, λex=270 nm), and various catalytic parameters such as Km, Vmax, and Kcat were calculated from Michaelis-Menten and Lineweaver-Burk plot. Efficiency of enzymatic hydrolysis was in the order of CalB > Si-MNPs@CalB > CalB@NF > Si-MNPs@CalB@NF, and reusability was much better with the enzyme immobilized on Si-MNPs than with the native enzyme. Enzymatic acylation was confirmed by the formation of benzyl benzoate (λex=229 nm) from benzoic anhydride (λex=240 nm), and efficiency exceeded 99% in all samples. Finally, enzymatic lipolysis was confirmed by the decomposition of olive oil to fatty acid such as oleic acid (λex=230 nm) using thin-layer chromatography (TLC), column chromatography, and proton nuclear magnetic resonance (1H -NMR). The lipases immobilized on Si-MNPs have the advantage of good thermal stability and reusability and are thus applicable to a wide range of industries.

Background. Sarcopenia and spinal cord injury (SCI) often coexist, but little is known about the associations of these two pathologies. This study aimed to assess the impact of SCI on muscle and bone mass and the correlations between clinical characteristics of SCI patients and sarcopenia. Methods: 136 patients diagnosed with SCI admitted to rehabilitation hospital were included in the study. The type and severity of injury (AIS), the level of spasticity (MAS), bone mineral density and Appendicular Lean Muscle Mass (ALM) were assessed. Sarcopenia was diagnosed according to the recommended EWGSOP2 cut-off points for ALM. Results: There was a statistically significant association between the ALM value and the gender, 75% of women developing sarcopenia. Among men, 42.9% had sarcopenia. Over 55% of AIS Grades A and B cases had sarcopenia. Mean age of sarcopenic patients was 35.8 years and they had multiple comorbidities. 51.6% of the patients with osteoporosis had sarcopenia. 69.7% of MAS level 0 cases had sarcopenia, while this was diagnosed in about 40% of patients with MAS levels 1-4. Conclusions: Gender, type of injury, presence of multiple comorbidities and age were directly associated with sarcopenia, while surprisingly, spasticity level and the presence of immobilization osteoporosis were not.

Immobilization of enzymes is a good field of study to expand the life of enzyme in and lowering the cost of the chemical processes such as separation processes. Urease is an important enzyme with medical and industrial applications. The aim of the present study is to prepare an immobilized urease on a strong cation exchange resin (Amberlite IR120 -Na) and study of its activity and stability. We monitored the liberation of Na ions in the collected fractions and searching for protein in the fractions as an indicators of immobilization by ion exchange phenomenon. Sodium is measured using atomic absorption spectroscopy techniques, while protein tested by Bradford’s method. Immobilized urease activity was evaluated by salicylate-hypochlorite method. The results indicated a complete immobilization of urease enzyme on the resin surface with reserving 92% of the activity of free enzyme. The immobilized urease enzyme on resin showed good stability and it has a 62% of its activity after 154 days of storage at room temperature. It is concluded that a new immobilized urease enzyme system is prepared with good enzyme activity and stability.

Enzyme immobilization is known to enhance polypeptide resistance towards environmental changes, particularly in terms of their stability. The Y509E mutant of β-xylosidase from Geobacil-lus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. In the immo-bilization by entrapment, besides observing a total loss of xylanase activity, it was found that chitosan concentration affects the process efficiency. Additionally, the maturation time of the spheres and the alkaline pH used to precipitate the spheres affect the β-xylosidase activity of the enzyme. In covalent bond immobilization, it was found that the pH during the modification with glutaraldehyde, as well as the subsequent protein binding, were key factors in preserving the dual enzymatic activity. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 U. g-1, respectively. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 40% and 88% of their activi-ty after being stored at 4 C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining a 92% of activity after ten cycles of re-use.

A new sustainable heterogenous catalyst for copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) was investigated. The preparation of the sustainable catalyst was carried out through the complexation reaction between the polysaccharide cellulose acetate backbone (CA) and copper(II) ions. The resulting complex [Cu(II)-CA] was fully characterized by using different spectroscopic methods such as FTIR, SEM, EDX, UV-Vis, and ICP analyses. The Cu(II)-CA complex exhibits a high activity in the CuAAC reaction for substituted alkynes and organic azides, leading to a selective synthesis of the corresponding 1,4-isomer 1,2,3-triazoles in water as solvent and working at room temperature. It is worth noting that this catalyst has several advantages from the sustainable chemistry point of view including no use of additives, biopolymer support, reactions carried out in water at room temperature, and easy recovery of the catalyst. These characteristics make it a potential candidate not only for the CuAAC reaction but also for other catalytic organic reactions.

Immobilization enables enzymes to be held in place so that they can be easily separated from the product when needed and can be used again. Conventional methods of immobilization include adsorption, encapsulation, entrapment, cross linking and covalent binding. However, conventional methods have several drawbacks including reduced stability, loss of biomolecules, less enzyme loading or activity and limited diffusion. The aim of this study is the evaluation of importance of nanomaterials for the immobilization of industrially important enzymes. Nano materials are now in trend for the immobilization of different enzymes due to their physiochemical properties. Gold nanoparticles, silver nanoparticles, nano diamonds, graphene, carbon nanotubes and others are used for immobilization. Among covalent and non-covalent immobilization of enzymes involving both single and multiwalled carbon nanotubes, non-covalent immobilization with functionalized carbon nanotubes is superior. Therefore, enzymes immobilized with nanomaterials possess greater stability, retention of catalytic activity and reusability of enzymes

A bacterial strain that could effectively degrade DEHP was isolated from the activated sludge and identified as Bacillus sp. by DNA sequencing. The biochemical degradation pathway of DEHP was further analyzed by GC-MS, and the results showed that DEHP was first decomposed into phthalates (DBP). Diuretic sylycol (DEP) was then generated, and phthalates (PA) were generated by a continuous de-ehelateization reaction. Phthalic acid (PA) was oxidized, dehydrogenated, and decarboxylated into protocatechins. Protocatechins enter the TCA cycle through orthotopic ring opening.　To enhance DEHP degradation, sodium alginate and calcium chloride were used as embedding and cross-linking materials, and the strain was immobilized. The immobilization conditions were optimized via an orthogonal experiment, and the results showed that the optimal immobilization conditions were SA mass fraction of 4%, CaCl2 mass fraction of 5%, ratio of bacteria to SA of 1:1, and the crosslinking time of 6 hours. The immobilized bacteria agent was further applied to MBR systems. The results showed that the removal rate of DEHP (5mg/L) in the system by immobilized bacteria was 91.9%, which is significantly higher than that of free bacteria. The 3, 4-dioxygenase gene and microbial community dynamics were analyzed by q-PCR and Illumina Miseq sequencing. The q-PCR results showed that the number of copies of 3, 4-dioxygenase gene in the immobilized system was significantly higher than that of free bacteria. Illumina Miseq sequencing results showed that Micromonospora, Rhodococcus, Bacteroides and Pseudomonas were the dominant generas in the MBR system. The analysis of bacterial community structure indicated that immobilization technology had a positive impact on the system stability. The results implied that this immobilized technique had potential applications in DEHP wastewater treatment.

The remediation of dredged marine sediments contaminated by heavy metals has drawn increasing attention worldly. The immobilization was regarded as a promising method to reduce adverse impacts on marine ecosystem. In this study, kaolinite and limestone were used as amendments to immobilize heavy metals (e.g. Zn, Pb and Cu) respectively in dredged marine sediments collected from the coastal zone adjacent to Tianjin Port in Bohai Bay. The sequential extraction procedure was applied to identify the mobility of heavy metals and further to evaluate the immobilization effect of amendments. The physical-chemical properties of sediments, such as pH, electrical conductivity (EC), salinity, and total organic carbon (TOC), were also measured to better understand their influence on heavy metals’ mobility. In addition, the compositions of clay minerals were also analyzed to identify the transformation process of minerals in the sediments. The results of sequential extraction procedure indicated that mobile fractions of heavy metals were converted into relatively stable fractions because of the two amendments. In addition, EC, salinity and TOC decreased moderately while no obvious variations of pH in the sediments were observed with the addition of the the kaolinite and the limestone. The percentage of montmorillonite decreased to minimum value while that of chlorite increased gradually during the experimental periods for 40 days probably due to complexation reaction. It was confirmed that both kaolinite and limestone can effectively reduce the mobility and bioavailability of heavy metals, particularly for Zn, generally, limestone has a better immobilization effect compared with kaolinite.

Extracellular enzymes catalyze biogeochemical reactions in soil, cycling carbon and nutrients in agricultural systems. Enzymes respond quickly to soil management, including organic amendment inputs, such as biochar, a charcoal-like solid byproduct of bioenergy production. In a previous agricultural field trial, a pine biochar amendment caused an approximately 40% decrease in the enzyme activities of β-glucosidase (BG) and phosphatase (PHOS). The large surface area of the pine biochar has the potential to sorb nutrients and other organic molecules. To test if sorption caused decreased enzyme activity, we used a laboratory assay to quantify the activity of two sorbed enzymes: BG and acid PHOS, involved in the cycling of carbon and phosphorous. The enzymes were incubated with three solid phases: (1) the high surface area pine biochar, (2) the agricultural soil, and (3) a low surface area grass biochar, for an additional comparison. We quantified the sorbed enzymes at pH 6, 7, and 8, using a Bradford protein assay, and measured the immobilized enzyme activities via high-throughput fluorometric analysis. After sorption onto pine biochar, detectable BG and PHOS activity levels dropped by over 95% relative to the soil, supporting direct sorption as one mechanism that reduces enzyme activity in biochar amended soil. This laboratory assay demonstrated that sorption could account for the lack of priming of native soil organic matter and changes in soil phosphorous cycling after pine biochar addition.

The synthesis of new collagen, chitosan and chitosan-collagen coated magnetic nanoparticles have been done. Two types of cross-linkers for polymer shell stabilization were used: glutaraldehyde (Gla) as a standard cross-linker and new one – squaric acid (SqA). Structure and morphology of prepared nanoparticles were characterized by ATR-FT IR, XRD and TEM analysis. The immobilization of lipase from Candida rugosa was performed on the nanoparticles surface. The amount of immobilized enzyme was quantified by the Bradford method. All of lipase-biopolymers coated nanoparticles were characterized with good activity recovery. A little hyperactivation of lipase immobilized on nanoparticles with SqA was observed. All of prepared lipase-immobilized nanoparticles were characterized with very good reusability.

The main goal of this research is to create biocompatible hydrogels using gelatin and a double cross-linking technique involving both covalent and ionic bonds to immobilize propolis. Covalent bonds are formed through Schiff base cross-links between protein-free amino groups (NH2) from the lysine residue and aldehyde groups (CHO) produced by oxidizing sodium alginate with NaIO4, while the ionic bonds are achieved using Mg2+ ions. Hydrogel films were obtained by varying the molar ratios -CHO/NH2 under different pH conditions (3.5 and 5.5). The presence of aldehyde groups in the oxidized sodium alginate (OSA) was confirmed using FTIR and NMR spectroscopy. The oxidation degree was monitored over 48 hours, and the influence of temperature was examined. Results showed that higher -CHO/-NH2 molar ratios led to increased conversion index values of NH2 groups, and a decrease in swelling degree values was observed in mediums with pH of 5.5 and 7.4. The encapsulation and release efficiency of propolis decreased with the increase of the hydrogel cross-linking degree. UV irradiation enhanced the antioxidant activity of both free and encapsulated propolis. These findings offer valuable insights into designing biocompatible hydrogels for propolis immobilization, with potential for controlled release in biomedical and pharmaceutical fields.

This study assessed the periodic fluctuations among microbial biomass Carbon (C), Nitrogen (N) and Phosphorus (P), and the consequences of variations in altitude and abiotic factors on the soil microbial biomass (C, N and P) in a temperate mixed-oak pine forest of Central Himalaya. This research was directed at three forest stands along an altitudinal gradient. Samples were collected in triplicates, seasonally from each selected site and microbial C, N and P were determined through the fumigation extraction method. Microbial biomass C, N and P decreased significantly (P<0.01, correlation coefficient -0.985, -0.963, -0.948, respectively) with increasing altitude, while the rainy season showed the highest values, and winter season revealed the least values. Microbial biomass C, N and P showed positive correlation with silt particles, water holding capacity, bulk density, porosity, soil moisture, organic C, total N and P, and negative correlations with sand particles and soil pH. The microbial biomass C showed strong associations with soil microbial N (r=0.80, P<0.01) and P (r=0.89, P<0.01) contents, while the soil microbial biomass N and P also showed strong positive correlation (r=0.92, P< 0.01). Soil microbial biomass was greatly influenced by the altitude and abiotic variables whereas, weakly by temporal variation. The microbial C: N ratio indicated that fertility of soil is influenced by the species assemblage. Our findings suggest that high microbial biomass and low C: N ratio during rainy season could be considered as a strategy to conserve nutrients by temperate mixed-oak pine forest ecosystem.

ω-Transaminases have been immobilized on macrocellular silica monoliths and used as heterogeneous biocatalysts in a continuous flow mode enantioselective transamination reaction. The support was prepared by a sol-gel method based on emulsion templating. The enzyme was immobilized on the structured silica monoliths both by adsorption, and by covalent grafting using amino-functionalized silica monoliths and glutaraldehyde as a coupling agent. A simple reactor set-up based on the use of a heat-shrinkable Teflon tube is presented and successfully used for the continuous flow kinetic resolution of a chiral amine, 4-bromo-α-methylbenzylamine. The porous structure of the supports ensures effective mass transfer and the reactor works in the plug flow regime without preferential flow paths. When immobilized in the monolith and used in the flow reactor, transaminases retain their activity and their enantioselectivity. The solid biocatalyst is also shown to be stable both on stream and during storage. These essential features pave the way to the successful development of an environmentally friendly process for chiral amines production.

Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans is successfully immobilized on 3-aminopropyltrimethoysilane functionalized MCF and SBA-15 silica supports using adsorption or covalently with glutaraldehyde or divinyl sulfone linkers. The best catalyst, AcmB on MCF linked covalently with glutaraldehyde, retains the specific activity of the homogenous enzyme while exhibiting a substantial increase of the operational stability. The immobilized enzyme was used continuously in the fed-batch reactor for 27 days, catalyzing 1,2-dehydrogenation of androst-4-en-3-one to androst-1,4-dien-3-one with a final yield of 29.9 mM (8.56 g/L) and 99% conversion. The possibility of reuse of the immobilized catalyst was also demonstrated and resulted with a doubling of the product amount compared to that in the reference homogenous reactor. Finally, it was shown that molecular oxygen from the air can efficiently be used as an electron acceptor either reoxidizing directly the enzyme or the reduced DCPIPH2. Keywords: 3-ketosteroid D1-dehydrogenase; KSTD; KSDH; AcmB; 1,2-dehydrogenation; cholest-4-en-3-one Δ1-dehydrogenase; enzyme immobilization, FAD-dependent enzymes; enzyme immobilization;

Almost 50 papers on surface plasmon resonance biosensors, published between 2016 and mid-2018, are reviewed. Papers concerning the determination of large particles such as vesicles, exosomes, cancer cells, living cells, stem cells and microRNA are excluded, as these are covered by a very recent review. The reviewed papers are categorized into five groups, depending on the degree of maturity of the reported solution: ranging from simple marker detection to clinical application of a previously developed biosensor. Instrumental solutions and details of biosensor construction are analyzed, including the chips, receptors and linkers used, as well as calibration strategies. Biosensors with a sandwich structure containing different nanoparticles are considered separately, as are SPR applications for investigating the interactions of biomolecules. An analysis is also made of the markers determined using the biosensors. Concluding, there is shown to be a growing number of SPR applications in the solution of real clinical problems.

Biofilm serves as the residence for bacteria, wherein bacteria attach to surfaces and produce extracellular polymeric substances, including proteins, polysaccharides, and extracellular DNA. In recent years, biofilm reactors have gained increasing significance, playing a crucial role, for instance, in immobilizing heavy metals from waterways. This paper employs a bibliographic study to analyze the most important keywords and organizations in the field of biofilm reactors, identifying recent significant papers. Additionally, the paper discusses the opportunities for the future development of biofilm reactors. Through this study, a better understanding of key factors in biofilm reactor research is achieved, providing valuable guidance for future studies and applications in this field.

L-asparaginase (ASNase) is an aminohydrolase currently used in the pharmaceutical and food industries. Enzyme immobilization is an exciting option for both applications, allowing a more straightforward recovery and increased stability. High surface area and customizable porosity make carbon xerogels (CXs) promising materials for ASNase immobilization. This work describes the influence of contact time, pH, and ASNase concentration on the immobilization yield (IY) and relative recovered activity (RRA) using Central Composite Design methodology. The most promising results were obtained using CX with an average pore size of 4 nm (CX-4), reaching IY and RRA of 100%. At the optimal conditions, the ASNase-CXs biocomposite was characterized and evaluated in terms of kinetic properties and operational, thermal and pH stabilities. The immobilized ASNase onto CX-4 retained 71% of its original activity after six continuous reaction cycles, showed a good thermal stability at 37 °C (RRA of 91% after 90 min) and was able to adapt to both acidic and alkaline environments. Finally, the results indicated a 3.9-fold increase in the immobilized ASNase affinity for the substrate, confirming the potential of CXs as a support for ASNase and as a cost-effective tool for subsequent use in the therapeutic and food sectors.

Nucleoside analogs are commonly used drugs for the treatment of cancer and viral infections. Nucleoside deoxyribosyltransferase (NDT) is one of the key enzymes required for the biosynthesis of deoxynucleotides. The feasibility of biochemical transformation processes is usually greatly dependent on the cost of biocatalysts. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it not only allows enzymes to be reused but can also improve their stability under several reaction conditions. In this work, histidine tags have been successfully fused to a Nucleoside deoxyribosyltransferase from Lactobacillus reuteri (LrDNT), which has been produced in a bench-scale reactor using a facile araBAD promoter-based protein expression system in an industrial microorganism, the E. coli BL21(DE3) strain. The overexpressed LrNDT was bound to a nickel-chelating agarose affinity chromatography medium via a one-step purification and immobilization process with a 99% yield, retaining about 97% of its initial activity. The optimal conditions for the immobilized LrNDT maintain high enzyme activity within the range of 20-45°C and pH 6.0-7.5. Finally, the immobilized enzyme reactor (IMER) of LrNDT was applied for the continuous-flow biosynthesis of 2'-deoxyadenosine and retained 90% of its activity after 16 hours, providing a theoretical basis for the industrial production of nucleoside analogs.

Protein–nanoparticle hybridization can ideally lead to novel biological entities characterized by emerging properties, which can sensibly differ from those of the parent components. Herein, the effect of ionic strength on the biological functions of recombinant, His-tagged spermine oxidase (i.e., SMOX) was studied for the first time. Moreover, SMOX was integrated to colloidal surface active maghemite nanoparticles (SAMNs) by direct self-assembly, leading to a biologically active nano-enzyme (i.e., SAMN@SMOX). The hybrid was subjected to an in-depth chemical-physical characterization highlighting that protein structure was perfectly preserved. The catalytic activity of the nanostructured hybrid (SAMN@SMOX) was assessed by extracting kinetics parameters using spermine as substrate and compared to the soluble enzyme as a function of ionic strength. Results revealed that the catalytic function was dominated by electrostatic interactions and that they were drastically modified upon hybridization with colloidal ɣ-Fe2O3. Noteworthy, the affinity of SMOX toward spermine was significantly higher for the nanohybrid at low salinity. The present study supports the vision of using protein–nanoparticle conjugation as a mean for modulating biological functions.

The development of a novel and simple inhibition biosensor array for detection of water pollutants based on immobilized bacteria is the main goal of this work. A series of electrochemical measurements (i.e. cyclic voltammograms) were carried out on screen-printed gold electrodes with three types of bacteria, namely Escherichia coli, Shewanella oneidensis, and Methylococcus capsulatus, immobilized via poly L-lysine. For comparison purposes, similar measurements were carried out on bacteria samples in solutions,; also optical measurements (fluorescence microscopy, optical density, and flow cytometry) were performed on the same bacteria in both liquid and immobilized forms. The study of the effect of heavy metal ions (lead), pesticides (atrazine) and petrochemicals (hexane) on DC electrochemical characteristics of immobilized bacteria revealed a possibility of pattern recognition of the above inhibition agents in aquatic environment.

Cadmium (Cd) contaminated soil is a serious environmental problem due to its effect on crop production, soil microbial community, and risk to human health. The commercial amendments are widely used in the immobilization of heavy metal-contaminated soil because of their cost-effectiveness, eco-friendliness, and community acceptance. This study is conducted to assess the effect of sole commercial amendments including bentonite (B), talc (T), activated carbon (AC), cornstarch (CS), and their combination on exchangeable Cd content in soil, Cd uptake by the plant, and soil microbial community. The results indicated that CS and its composites (T+CS and B+CS) showed the highest effect on reducing Cd mobility in soil (up to 10% in CS), decreasing the Cd uptake by lettuce (up to 78% in T+CS), and increasing the microbial community in soil compared to the control. Furthermore, results from Biolog Ecoplate and realtime-Polymerase chain reaction indicated that the mixture amendments (B+CS and T+CS) showed the highest effect on the microbial community by increasing soil microbial population maximum of 3.81-fold difference (in B+CS) compared to the control. The results indicated that applying composite amendments provided an efficient option for immobilizing Cd in soil, reducing the Cd uptake by plants, and increasing microbial community.

A portable urea sensor for use in the fast flow condition was fabricated using porous polytetrafluoroethylene (PTFE) membranes coated with amine-functionalized parylene, parylene-A, by vapor deposition. To generate a specific electrochemical sensor signal from urea, the urea-hydrolyzing enzyme urease was immobilized on the parylene-A-coated PTFE membranes via chemical crosslinking using glutaraldehyde. The urease-immobilized membranes were assembled in a polydimethylsiloxane (PDMS) fluidic chamber, and a screen-printed carbon three-electrode system was used for electrochemical measurements. The success of urease immobilization was confirmed using fluorescence microscopy, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The optimum concentration of urease for immobilization on the parylene-A-coated PTFE membranes was determined to be 48 mg/mL, and the optimum number of membranes in the PDMS chamber was found to be 8. Using these optimized conditions, we fabricated the urea biosensor and monitored urea samples under various flow rates ranging from 0.5 to 10 mL/min in the flow condition using chronoamperometry. To test the applicability of the sensor for physiological samples, we used it for monitoring urea concentration in the waste peritoneal dialysate of a patient with chronic renal failure, at a flow rate of 0.5 mL/min.

The dye effluent is usually difficult to be degraded by conventional wastewater treatment in leather industry. In order to develop efficient and cost-effective treatment methods, we evaluate the effect of white-rot fungus immobilization for dye decolorisation in this paper. The Phanerochaete chrysosporium BKM-F-1767 was used for immobilization. This research found that the white-rot fungus immobilization had an obviously decolorisation effect in dye wastewater treatment, and plant carriers such as sorghum stalk and corn cob were helpful to the growth of Phanerochaete chrysosporium in white-rot fungus immobilization. Due to the stability and recyclability, the white-rot fungus co-immobilization was considered as the most suitable treatment for decolorisation of dye effluent which enjoyed the advantages of both adsorption immobilization and entrapment immobilization. Furthermore, the dye decolorisation evaluation was carried out to find the most suitable carrier for co-immobilization, and it found that sorghum stalk - calcium-alginate gel spherical particle (SS-CGPB) has better decolorisation effect than corn cobs - calcium-alginate gel spherical particle (CC-CGPB), and the dye decolorisation rate was 86.77%. After 5 cycles, the dye decolorisation rate was 85.87% which indicated the SS-CGPB preserved functional integrity successfully. By further analyzing the biodegradation process with white-rot fungus immobilization, the intermediate products were observed and the degradation pathway of acid golden yellow dye molecular was proposed. The results showed that the C-N single bonds attached to the central benzene in the dye molecule were attacked and destroyed in white-rot fungus co-immobilization treatment, thus the structure of dye molecule could be successfully degraded into small molecules which would be more easily treated by conventional treatment methods. Therefore, the white rot fungus co-immobilization might be appropriate for pre-treatment as an important biotechnology for the advanced treatment of dye effluent.

Type-1 Diabetes Mellitus (T1D) onset and development is closely linked with imbalance of gut microbiome, while supplementation with probiotics may result in positive alterations in gut microbiome composition. In the present study, the effect of a dietary intervention including free or immobilized cells of the presumptive probiotic Pediococcus acidilactici ORE5 on Corinthian currants, a food with beneficial impact in the condition of diabetes, was investigated. Male Wistar rats were studied which were divided into 4 groups: healthy animals, which received the free (H_FP) or the immobilized cells (H_IPC), and diabetic animals, which received the free (D_FP) or the immobilized cells (D_IPC), for 4 weeks (109 cfu/day, in all groups). At the end of the dietary intervention, D_IPC group exerted lower concentration of the inflammatory cytokine IL-1b compared to D_FP. Consumption of immobilized on Corinthian currants P. acidilactici ORE5 cells by diabetic animals led to increased loads of fecal lactobacilli and lower levels of Enterobacteriaceae, coliforms, and E. coli, while Actinobacteria phylum, Akkermansia, and Bifidobacterium genera abundances were increased, and fecal lactic acid was elevated. Overall, the results of the present research demonstrated that functional ingredients can ameliorate gut dysbiosis present in T1D and could be used to design dietary patterns aiming at T1D management. However, well-designed clinical trials are necessary, in order to confirm the beneficial effects in humans.

Biochar application to the soil can improve soil quality and nutrient leaching loss. Recent studies have reported that surficial application of biochar to stored swine manure can reduce emissions of odorous compounds and reduce the volatilization loss of ammonia. Our working hypothesis was that the biochar-treated manure application to the soil would decrease nutrient leaching from manure and increase plant-available nutrients. The study objectives were to evaluate the impact of biochar-treated swine manure on soil total C, N, and other major and minor nutrients. Three biochars (i) neutral pH red-oak (RO), (ii) highly alkaline autothermal corn (Zea mays) stover (HAP), and (iii) mild acidic Fe-treated autothermal corn stover (HAPE) were incubated with swine manure for a month. The biochar-manure mixture was applied in triplicate to soil columns with application rate determined by the P₂O₅-P content in manure or manure-biochar mixtures after the incubation. The ammonium (NH₄⁺), nitrate (NO₃^-), and reactive P concentrations in soil column leachates were recorded for eight leaching events. Soil properties and plant-available nutrients were compared between treatments and control manure & soil. Manure-(HAP&HAPE) biochar treatments significantly increased soil organic matter (OM) and increased soil total C, N, and improved soil bulk density. Concentrations of KCl-extractable NH₄⁺ and NO₃^- significantly increased in HAPE column leachates during this 4-week study and in the soil after the experiment. A significant reduction in soil Mehlich3 Cu was also observed for the manure-HAPE mixture compared with the control. Overall, the manure-biochar incubation enabled biochar to sorb nutrients from manure, and the subsequent manure-biochar mixture application to soil improved soil quality and plant nutrient availability in comparison to conventional manure application to soil. This proof-of-the-concept study suggests that biochars could be used to solve both environmental and agronomic challenges and further improve the sustainability of animal and crop production agriculture.

The problem of effective immobilization of liquid radioactive waste (LRW) is key to the successful development of nuclear energy. The possibility of using magnesium potassium phosphate (MKP) compound for LRW immobilization on the example of nitric acid solutions containing actinides and rare earth elements (REE), including high level waste (HLW) surrogate solution is considered in the research work. Under the study of phase composition and structure of the MKP compounds obtained by the XRD and SEM methods, it was established that the compounds are composed of crystalline phases - analogues of natural phosphate minerals (struvite, metaankoleite). The hydrolytic stability of the compounds was determined according to the semi-dynamic test GOST R 52126-2003. Low leaching rates of radionuclides from the compound are established, including a differential leaching rate of ²³⁹Pu and ²⁴¹Am - 3.5 × 10^-7 and 5.3 × 10^-7 g/(cm²∙day). As a result of the research work it was concluded that the MKP compound is promising for LRW immobilization and can become an alternative material combining the advantages of easy implementation of the technology like cementation and the high physical and chemical stability corresponding to a glass-like compound.

Pandemic human influenza detections are dependent on complex, time-consuming DNA molecular amplifications that require sophisticated PCR electrophoresis, cell culture amplification and complex spectrophotometric absorption techniques. In this study, simplified rapid electrochemical sensing methods were sought via a remote cyclic voltammetry and stripping amplifier circuit using our workstation systems. A sensing probe was prepared using human DNA and a Covid 19 antigen immobilized antibody sensing amplifier with rapid accumulation time of 30 sec. Optimal experimentation was performed using a telematics WiFi wearable circuit equipped with a bioelectrochemical analyzer-2 amplifier three electrode system. Diagnostic accumulation voltammetry and cyclic redox scanning were attempted at -2.0V~2.0V potential windows and 0.2V/Sec scan rate. Anodic and cathodic real time assay was performed using saliva swab from female patients that tested negative and positive. Diagnostic results are expected to be useful for non-contact analysis and for rapid personal testing anytime, anywhere.

As a consequence of intense industrialization in the last few decades, the amount of agro-industrial wastes has increasing, where new forms of valorization are crucial. In this work, 5 residual biomasses from Maranhão (Brazil) were investigted as supports for immobilization of lipase from Thermomyces lanuginosus (TLL). The new biocatalysts BM-TLL (babaçu mesocarp) and RH-TLL (rice husk) showed immobilization efficiencies &gt;98% and hydrolytic activities of 5,331 U.g-1 and 4.608 U. g-1 respectively against 142 U. g-1 by Lipozyme® TL IM. High esterification activities were also found, with 141.4 U.g-1 and 396.4 U.g-1 from BM-TLL and RH-TLL against 113.5 U.g-1 by TL IM. Results of porosimetry, SEM and BET demonstrated BM and RH supports are mesoporous materials with large hydrophobic area, allowing a mixture of hydrophobic adsorption and confinement, resulting in hyperactivation of TLL. These biocatalysts were applied in the production of hexyl laurate, where RH-TLL was able to generate 94% conversion in 4 h. Desorption with Triton X-100 and NaCl confirmed that new biocatalysts were more efficient with 5 times less protein than commercial TL IM. All results demonstrated that residual biomass was able to produce robust and stable biocatalysts containing immobilized TLL with better results than commercial preparations.

The anode of a microbial fuel cell (MFC) was formed on a graphite electrode and immobilized Gluconobacter oxydans VKM-1280 bacterial cells. Immobilization was performed in chitosan, poly(vinyl alcohol) or N-vinylpyrrolidone-modified poly(vinyl alcohol). Ethanol was used as substrate. The anode was modified using multiwalled carbon nanotubes. The aim of the modification was to create a conductive network between cell lipid membranes, containing exposed PQQ-dependent alcoholdehydrogenases, and the electrode to facilitate electron transfer in the system. The bioelectrochemical characteristics of modified anodes at various cell/polymer ratios were assessed via current density, power density, polarization curves and impedance spectres. MFCs based on chitosan at a matrix/cell volume ratio of 5:1 produced maximal power characteristics of the system (8.3 μW/cm2) at a minimal resistance (1111 Ohm cm2). Modification of the anode by multiwalled carbon nanotubes led to a slight decrease of internal resistance (down to 1078 Ohm cm2) and to an increase of generated power density up to 10.6 μW/cm2. We explored the possibility of accumulating electric energy from an MFC on a 6,800-μF capacitor via a boost converter. Generated voltage was increased from 0.3 V up to 3.2 V. Accumulated energy was used to power a Clark-type biosensor and a bluetooth transmitter with three sensors, a miniature electric motor and a light-emitting diode.

The development of rapid analysis of human serum for the presence of allergen-specific IgE is currently important. Consequently, we developed two types of 3D protein biochips. The first one is a 3D hydrogel biochip containing hydrogel droplets with protein molecules (allergens, immunoglobulins and others). These droplets are disposed on elements consisting of short polymer brushes grafting from a surface of polybutylene terephthalate polymer. The immobilization of proteins was induced by short-wave UV radiation. On such a biochip, the kinetics of allergen-sIgE complex formation reached 70% of saturation for 6 hours. Also we developed a 3D brush microchip containing on the surface of a polyethylene terephthalate polymer the brush elements with protein molecules covalently immobilized by opening oxirane cycles by amino and thiol nucleophilic groups contained in proteins. In the case of 3D brush microchip the kinetics of allergen-sIgE complex formation reached 100% of saturation for 3 hours, and fluorescent signals were 2-3 times higher than those of the 3D hydrogel biochip. Thus, the comparative analysis revealed that 3D brush biochips are more useful for further studies of protein-protein interactions than 3D hydrogel ones.

A variational analysis of dispersion medium (electrolytes and surfactants) was carried out for the best nucleotide interactions with the presence of nanoparticles and the ability to formation of homogeneous coatings upon drying / lyophilization. Using UV-spectroscopy and transmission electron microscopy, the processes of immobilization of nucleotides on the surface of nanoparti-cles of the composition ZrO2-3mol% Y2O3 (YSZ) in the presence of various dispersing agents were investigated. Promising dispersing agents were established, the optimal composition of suspen-sions based on DNA nucleotides and YSZ nanoparticles, which is potentially suitable for adaptive technologies of bionanoelectronics, was selected. The aim of the work is to obtain and study functional media for molecular electronics based on heterojunctions biological molecule – wide-gap dielectric.

A variational analysis of dispersion medium (electrolytes and surfactants) was carried out for the best nucleotide interactions with the presence of nanoparticles and the ability to formation of homogeneous coatings upon drying / lyophilization. Using UV-spectroscopy and transmission electron microscopy, the processes of immobilization of nucleotides on the surface of nanoparti-cles of the composition ZrO2-3mol% Y2O3 (YSZ) in the presence of various dispersing agents were investigated. Promising dispersing agents were established, the optimal composition of suspen-sions based on DNA nucleotides and YSZ nanoparticles, which is potentially suitable for adaptive technologies of bionanoelectronics, was selected. The aim of the work is to obtain and study functional media for molecular electronics based on heterojunctions biological molecule – wide-gap dielectric.

Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads.