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An improved and convenient ninhydrin assay for aminoacylase activity measurements was developed using the commercial EZ Nin™ reagent. Alternative reagents from literature were also evaluated and compared. The addition of DMSO to the reagent enhanced the solubility of Ruhemann's purple (RP). Furthermore, we found that the use of a basic, aqueous buffer enhances stability of RP. An acidic protocol for the quantification of lysine was developed by addition of glacial acetic acid. The assay allows for parallel processing in a 96-well format with measurements microtiter plates.
Acetoin and diacetyl have a major impact on the flavor of alcoholic beverages such as wine or beer. Therefore, their measurement is important during the fermentation process. Until now, gas chromatographic techniques have typically been applied; however, these require expensive laboratory equipment and trained staff, and do not allow for online monitoring. In this work, a capacitive electrolyte–insulator–semiconductor sensor modified with tobacco mosaic virus (TMV) particles as enzyme nanocarriers for the detection of acetoin and diacetyl is presented. The enzyme acetoin reductase from Alkalihalobacillus clausii DSM 8716ᵀ is immobilized via biotin–streptavidin affinity, binding to the surface of the TMV particles. The TMV-assisted biosensor is electrochemically characterized by means of leakage–current, capacitance–voltage, and constant capacitance measurements. In this paper, the novel biosensor is studied regarding its sensitivity and long-term stability in buffer solution. Moreover, the TMV-assisted capacitive field-effect sensor is applied for the detection of diacetyl for the first time. The measurement of acetoin and diacetyl with the same sensor setup is demonstrated. Finally, the successive detection of acetoin and diacetyl in buffer and in diluted beer is studied by tuning the sensitivity of the biosensor using the pH value of the measurement solution.
A capacitive electrolyte-insulator-semiconductor (EISCAP) biosensor modified with Tobacco mosaic virus (TMV) particles for the detection of acetoin is presented. The enzyme acetoin reductase (AR) was immobilized on the surface of the EISCAP using TMV particles as nanoscaffolds. The study focused on the optimization of the TMV-assisted AR immobilization on the Ta 2 O 5 -gate EISCAP surface. The TMV-assisted acetoin EISCAPs were electrochemically characterized by means of leakage-current, capacitance-voltage, and constant-capacitance measurements. The TMV-modified transducer surface was studied via scanning electron microscopy.
Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO₂-Ta₂O₅ layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1–3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta₂O₅-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.
This study addresses a proof-of-concept experiment with a biocompatible screen-printed carbon electrode deposited onto a biocompatible and biodegradable substrate, which is made of fibroin, a protein derived from silk of the Bombyx mori silkworm. To demonstrate the sensor performance, the carbon electrode is functionalized as a glucose biosensor with the enzyme glucose oxidase and encapsulated with a silicone rubber to ensure biocompatibility of the contact wires. The carbon electrode is fabricated by means of thick-film technology including a curing step to solidify the carbon paste. The influence of the curing temperature and curing time on the electrode morphology is analyzed via scanning electron microscopy. The electrochemical characterization of the glucose biosensor is performed by amperometric/voltammetric measurements of different glucose concentrations in phosphate buffer. Herein, systematic studies at applied potentials from 500 to 1200 mV to the carbon working electrode (vs the Ag/AgCl reference electrode) allow to determine the optimal working potential. Additionally, the influence of the curing parameters on the glucose sensitivity is examined over a time period of up to 361 days. The sensor shows a negligible cross-sensitivity toward ascorbic acid, noradrenaline, and adrenaline. The developed biocompatible biosensor is highly promising for future in vivo and epidermal applications.
Miniaturized electrolyte–insulator–semiconductor capacitors (EISCAPs) with ultrathin gate insulators have been studied in terms of their pH-sensitive sensor characteristics: three different EISCAP systems consisting of Al–p-Si–Ta2O5(5 nm), Al–p-Si–Si3N4(1 or 2 nm)–Ta2O5 (5 nm), and Al–p-Si–SiO2(3.6 nm)–Ta2O5(5 nm) layer structures are characterized in buffer solution with different pH values by means of capacitance–voltage and constant capacitance method. The SiO2 and Si3N4 gate insulators are deposited by rapid thermal oxidation and rapid thermal nitridation, respectively, whereas the Ta2O5 film is prepared by atomic layer deposition. All EISCAP systems have a clear pH response, favoring the stacked gate insulators SiO2–Ta2O5 when considering the overall sensor characteristics, while the Si3N4(1 nm)–Ta2O5 stack delivers the largest accumulation capacitance (due to the lower equivalent oxide thickness) and a higher steepness in the slope of the capacitance–voltage curve among the studied stacked gate insulator systems.
Nanoparticles are recognized as highly attractive tunable materials for designing field-effect biosensors with enhanced performance. In this work, we present a theoretical model for electrolyte-insulator-semiconductor capacitors (EISCAP) decorated with ligand-stabilized charged gold nanoparticles. The charged AuNPs are taken into account as additional, nanometer-sized local gates. The capacitance-voltage (C–V) curves and constant-capacitance (ConCap) signals of the AuNP-decorated EISCAPs have been simulated. The impact of the AuNP coverage on the shift of the C–V curves and the ConCap signals was also studied experimentally on Al–p-Si–SiO₂ EISCAPs decorated with positively charged aminooctanethiol-capped AuNPs. In addition, the surface of the EISCAPs, modified with AuNPs, was characterized by scanning electron microscopy for different immobilization times of the nanoparticles.
Frequency mixing magnetic detection (FMMD) has been explored for its applications in fields of magnetic biosensing, multiplex detection of magnetic nanoparticles (MNP) and the determination of core size distribution of MNP samples. Such applications rely on the application of a static offset magnetic field, which is generated traditionally with an electromagnet. Such a setup requires a current source, as well as passive or active cooling strategies, which directly sets a limitation based on the portability aspect that is desired for point of care (POC) monitoring applications. In this work, a measurement head is introduced that involves the utilization of two ring-shaped permanent magnets to generate a static offset magnetic field. A steel cylinder in the ring bores homogenizes the field. By variation of the distance between the ring magnets and of the thickness of the steel cylinder, the magnitude of the magnetic field at the sample position can be adjusted. Furthermore, the measurement setup is compared to the electromagnet offset module based on measured signals and temperature behavior.
In this study, an online multi-sensing platform was engineered to simultaneously evaluate various process parameters of food package sterilization using gaseous hydrogen peroxide (H₂O₂). The platform enabled the validation of critical aseptic parameters. In parallel, one series of microbiological count reduction tests was performed using highly resistant spores of B. atrophaeus DSM 675 to act as the reference method for sterility validation. By means of the multi-sensing platform together with microbiological tests, we examined sterilization process parameters to define the most effective conditions with regards to the highest spore kill rate necessary for aseptic packaging. As these parameters are mutually associated, a correlation between different factors was elaborated. The resulting correlation indicated the need for specific conditions regarding the applied H₂O₂ gas temperature, the gas flow and concentration, the relative humidity and the exposure time. Finally, the novel multi-sensing platform together with the mobile electronic readout setup allowed for the online and on-site monitoring of the sterilization process, selecting the best conditions for sterility and, at the same time, reducing the use of the time-consuming and costly microbiological tests that are currently used in the food package industry.
This work introduces a novel method for the detection of H₂O₂ vapor/aerosol of low concentrations, which is mainly applied in the sterilization of equipment in medical industry. Interdigitated electrode (IDE) structures have been fabricated by means of microfabrication techniques. A differential setup of IDEs was prepared, containing an active sensor element (active IDE) and a passive sensor element (passive IDE), where the former was immobilized with an enzymatic membrane of horseradish peroxidase that is selective towards H₂O₂. Changes in the IDEs’ capacitance values (active sensor element versus passive sensor element) under H₂O₂ vapor/aerosol atmosphere proved the detection in the concentration range up to 630 ppm with a fast response time (<60 s). The influence of relative humidity was also tested with regard to the sensor signal, showing no cross-sensitivity. The repeatability assessment of the IDE biosensors confirmed their stable capacitive signal in eight subsequent cycles of exposure to H₂O₂ vapor/aerosol. Room-temperature detection of H₂O₂ vapor/aerosol with such miniaturized biosensors will allow a future three-dimensional, flexible mapping of aseptic chambers and help to evaluate sterilization assurance in medical industry.
Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities of potential heterologous amylases, however, cannot be quantified directly from the supernatant due to the high background activity of native α-amylase. This activity is caused by the gene products of amyA, amyB, and amyC. In this study, an in vitro CRISPR/Cas9 system was established in A. oryzae to delete these genes simultaneously. First, pyrG of A. oryzae NSAR1 was mutated by exploiting NHEJ to generate a counter-selection marker. Next, all amylase genes were deleted simultaneously by co-transforming a repair template carrying pyrG of Aspergillus nidulans and flanking sequences of amylase gene loci. The rate of obtained triple knock-outs was 47%. We showed that triple knockouts do not retain any amylase activity in the supernatant. The established in vitro CRISPR/Cas9 system was used to achieve sequence-specific knock-in of target genes. The system was intended to incorporate a single copy of the gene of interest into the desired host for the development of screening methods. Therefore, an integration cassette for the heterologous Fpi amylase was designed to specifically target the amyB locus. The site-specific integration rate of the plasmid was 78%, with exceptional additional integrations. Integration frequency was assessed via qPCR and directly correlated with heterologous amylase activity. Hence, we could compare the efficiency between two different signal peptides. In summary, we present a strategy to exploit CRISPR/Cas9 for gene mutation, multiplex knock-out, and the targeted knock-in of an expression cassette in A. oryzae. Our system provides straightforward strain engineering and paves the way for development of fungal screening systems.
Background
Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes.
Results
We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system.
Conclusion
Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.
Herein, fibroin, polylactide (PLA), and carbon are investigated for their suitability as biocompatible and biodegradable materials for amperometric biosensors. For this purpose, screen-printed carbon electrodes on the biodegradable substrates fibroin and PLA are modified with a glucose oxidase membrane and then encapsulated with the biocompatible material Ecoflex. The influence of different curing parameters of the carbon electrodes on the resulting biosensor characteristics is studied. The morphology of the electrodes is investigated by scanning electron microscopy, and the biosensor performance is examined by amperometric measurements of glucose (0.5–10 mM) in phosphate buffer solution, pH 7.4, at an applied potential of 1.2 V versus a Ag/AgCl reference electrode. Instead of Ecoflex, fibroin, PLA, and wound adhesive are tested as alternative encapsulation compounds: a series of swelling tests with different fibroin compositions, PLA, and Ecoflex has been performed before characterizing the most promising candidates by chronoamperometry. Therefore, the carbon electrodes are completely covered with the particular encapsulation material. Chronoamperometric measurements with H2O2 concentrations between 0.5 and 10 mM enable studying the leakage current behavior.
The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465ᵀ and Alkalibacillus haloalkaliphilus DSM 5271ᵀ and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976ᵀ served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0–12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.
Hydrogen peroxide (H₂O₂), a strong oxidizer, is a commonly used sterilization agent employed during aseptic food processing and medical applications. To assess the sterilization efficiency with H₂O₂, bacterial spores are common microbial systems due to their remarkable robustness against a wide variety of decontamination strategies. Despite their widespread use, there is, however, only little information about the detailed time-resolved mechanism underlying the oxidative spore death by H₂O₂. In this work, we investigate chemical and morphological changes of individual Bacillus atrophaeus spores undergoing oxidative damage using optical sensing with trapping Raman microscopy in real-time. The time-resolved experiments reveal that spore death involves two distinct phases: (i) an initial phase dominated by the fast release of dipicolinic acid (DPA), a major spore biomarker, which indicates the rupture of the spore’s core; and (ii) the oxidation of the remaining spore material resulting in the subsequent fragmentation of the spores’ coat. Simultaneous observation of the spore morphology by optical microscopy corroborates these mechanisms. The dependence of the onset of DPA release and the time constant of spore fragmentation on H₂O₂ shows that the formation of reactive oxygen species from H₂O₂ is the rate-limiting factor of oxidative spore death.
Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.
Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications.
Lead and nickel, as heavy metals, are still used in industrial processes, and are classified as “environmental health hazards” due to their toxicity and polluting potential. The detection of heavy metals can prevent environmental pollution at toxic levels that are critical to human health. In this sense, the electrolyte–insulator–semiconductor (EIS) field-effect sensor is an attractive sensing platform concerning the fabrication of reusable and robust sensors to detect such substances. This study is aimed to fabricate a sensing unit on an EIS device based on Sn₃O₄ nanobelts embedded in a polyelectrolyte matrix of polyvinylpyrrolidone (PVP) and polyacrylic acid (PAA) using the layer-by-layer (LbL) technique. The EIS-Sn₃O₄ sensor exhibited enhanced electrochemical performance for detecting Pb²⁺ and Ni²⁺ ions, revealing a higher affinity for Pb²⁺ ions, with sensitivities of ca. 25.8 mV/decade and 2.4 mV/decade, respectively. Such results indicate that Sn₃O₄ nanobelts can contemplate a feasible proof-of-concept capacitive field-effect sensor for heavy metal detection, envisaging other future studies focusing on environmental monitoring.