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Enzyme-catalyzed reactions have been designed to mimic various Boolean logic gates in the general framework of unconventional biomolecular computing. While some of the logic gates, particularly OR, AND, are easy to realize with biocatalytic reactions and have been reported in numerous publications, some other, like NXOR, are very challenging and have not been realized yet with enzyme reactions. The paper reports on a novel approach to mimicking the NXOR logic gate using the bell-shaped enzyme activity dependent on pH values. Shifting pH from the optimum value to the acidic or basic values by using acid or base inputs (meaning 1,0 and 0,1 inputs) inhibits the enzyme reaction, while keeping the optimum pH (assuming 0,0 and 1,1 input combinations) preserves a high enzyme activity. The challenging part of the present approach is the selection of an enzyme with a well-demonstrated bell-shape activity dependence on the pH value. While many enzymes can satisfy this condition, we selected pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase as this enzyme has the optimum pH center-located on the pH scale allowing the enzyme activity change by the acidic and basic pH shift from the optimum value corresponding to the highest activity. The present NXOR gate is added to the biomolecular “toolbox” as a new example of Boolean logic gates based on enzyme reactions.
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.
The incorporation of nanomaterials that are biocompatible with different types of biological compounds has allowed the development of a new generation of biosensors applied especially in the biomedical field. In particular, the integration of film-based nanomaterials employed in field-effect devices can be interesting to develop biosensors with enhanced properties. In this paper, we studied the fabrication of sensitive nanofilms combining ZnO nanocrystals and carbon nanotubes (CNTs), prepared by means of the layer-by-layer (LbL) technique, in a capacitive electrolyte-insulator-semiconductor (EIS) structure for detecting glucose and urea. The ZnO nanocrystals were incorporated in a polymeric matrix of poly(allylamine) hydrochloride (PAH), and arranged with multi-walled CNTs in a LbL PAH-ZnO/CNTs film architecture onto EIS chips. The electrochemical characterizations were performed by capacitance–voltage and constant capacitance measurements, while the morphology of the films was characterized by atomic force microscopy. The enzymes glucose oxidase and urease were immobilized on film’s surface for detection of glucose and urea, respectively. In order to obtain glucose and urea biosensors with optimized amount of sensitive films, we investigated the ideal number of bilayers for each detection system. The glucose biosensor showed better sensitivity and output signal for an LbL PAH-ZnO/CNTs nanofilm with 10 bilayers. On the other hand, the urea biosensor presented enhanced properties even for the first bilayer, exhibiting high sensitivity and output signal. The presence of the LbL PAH-ZnO/CNTs films led to biosensors with better sensitivity and enhanced response signal, demonstrating that the adequate use of nanostructured films is feasible for proof-of-concept biosensors with improved properties that may be employed for biomedical applications.