Wiley
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.
A concept for a new generation of an integrated multi-functional biosensor/actuator system is developed, which is based on biomolecular logic principles. Such a system is expected to be able to detect multiple biochemical input signals simultaneously and in real-time and convert them into electrical output signals with logical operations such as OR, AND, etc. The system can be designed as a closed-loop drug release device triggered by an enzyme logic gate, while the release of the drug induced by the actuator at the required dosage and timing will be controlled by an additional drug sensor. Thus, the system could help to make an accurate and specific diagnosis. The presented concept is exemplarily demonstrated by using an enzyme logic gate based on a glucose/glucose oxidase system, a temperature-responsive hydrogel mimicking the actuator function and an insulin (drug) sensor. In this work, the results of functional testing of individual amperometric glucose and insulin sensors as well as an impedimetric sensor for the detection of the hydrogel swelling/shrinking are presented.
We present a sensor concept based on copper(II)oxide (CuO) nanofibres for the detection of hydrogen peroxide (H2O2) vapour in the percent per volume (% v/v) range. The fibres were produced by using the electrospinning technique. To avoid water condensation in the pores, the fibres were initially modified by an exposure to H2S to get an enclosed surface. By a thermal treatment at 350 °C the fibres were oxidised back to CuO. Thereby, the visible pores disappear which was verified by SEM analysis. The fibres show a decrease of resistance with increasing H2O2 concentration which is due to the fact that hydrogen peroxide is an oxidising gas and CuO a p-type semiconductor. The sensor shows a change of resistance within the minute range to the exposure until the maximum concentration of 6.9% v/v H2O2. At operating temperatures below 450 °C the corresponding sensor response to a concentration of 4.1% v/v increases. The sensor shows a good reproducibility of the signal at different measurements. CuO seems to be a suitable candidate for the detection of H2O2 vapour at high concentrations.
Resistance behaviour of the sensor under exposure to H2O2 vapours between 2.3 and 6.9% v/v at an operating temperature of 450 °C.
The integration of biomolecular logic principles with electronic transducers allows designing novel digital biosensors with direct electrical output, logically triggered drug-release, and closed-loop sense/act/treat systems. This opens new opportunities for advanced personalized medicine in the context of theranostics. In the present work, we will discuss selected examples of recent developments in the field of interfacing enzyme logic gates with electrodes and semiconductor field-effect devices. Special attention is given to an enzyme OR/Reset logic gate based on a capacitive field-effect electrolyte-insulator-semiconductor sensor modified with a multi-enzyme membrane. Further examples are a digital adrenaline biosensor based on an AND logic gate with binary YES/NO output and an integrated closed-loop sense/act/treat system comprising an amperometric glucose sensor, a hydrogel actuator, and an insulin (drug) sensor.