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Photoelectrochemical (PEC) biosensors are a rather novel type of biosensors thatutilizelighttoprovideinformationaboutthecompositionofananalyte,enablinglight-controlled multi-analyte measurements. For enzymatic PEC biosensors,amperometric detection principles are already known in the literature. In con-trast, there is only a little information on H+-ion sensitive PEC biosensors. Inthis work, we demonstrate the detection of H+ions emerged by H+-generatingenzymes, exemplarily demonstrated with penicillinase as a model enzyme on atitanium dioxide photoanode. First, we describe the pH sensitivity of the sensorand study possible photoelectrocatalytic reactions with penicillin. Second, weshow the enzymatic PEC detection of penicillin.
Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.
Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T. The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0–12.0 and temperature 20–80 °C, optimally at pH 9.0–9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2O2 for 1 h while stimulated at 1% (v/v) H2O2. Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future.
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.
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.
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.
In this work, the bioabsorbable materials, namely fibroin, polylactide acid (PLA), magnesium and magnesium oxide are investigated for their application as transient, resistive temperature detectors (RTD). For this purpose, a thin-film magnesium-based meander-like electrode is deposited onto a flexible, bioabsorbable substrate (fibroin or PLA) and encapsulated (passivated) by additional magnesium oxide layers on top and below the magnesium-based electrode. The morphology of different layered RTDs is analyzed by scanning electron microscopy. The sensor performance and lifetime of the RTD is characterized both under ambient atmospheric conditions between 30°C and 43°C, and wet tissue-like conditions with a constant temperature regime of 37°C. The latter triggers the degradation process of the magnesium-based layers. The 3-layers RTDs on a PLA substrate could achieve a lifetime of 8.5 h. These sensors also show the best sensor performance under ambient atmospheric conditions with a mean sensitivity of 0.48 Ω/°C ± 0.01 Ω/°C.