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Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC–MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed.
In this paper, carbon nanotubes (CNTs) were incorporated in penicillinase-phospholipid Langmuir and Langmuir–Blodgett (LB) films to enhance the enzyme catalytic properties. Adsorption of the penicillinase and CNTs at dimyristoylphosphatidic acid (DMPA) monolayers at the air–water interface was investigated by surface pressure–area isotherms, vibrational spectroscopy, and Brewster angle microscopy. The floating monolayers were transferred to solid supports through the LB technique, forming mixed DMPA-CNTs-PEN films, which were investigated by quartz crystal microbalance, vibrational spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV–vis spectroscopy and the feasibility of the supramolecular device nanostructured as ultrathin films were essayed in a capacitive electrolyte–insulator–semiconductor (EIS) sensor device. The presence of CNTs in the enzyme–lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks, showing increased values of activity. Viability as penicillin sensor was demonstrated with capacitance/voltage and constant capacitance measurements, exhibiting regular and distinctive output signals over all concentrations used in this work. These results may be related not only to the nanostructured system provided by the film, but also to the synergism between the compounds on the active layer, leading to a surface morphology that allowed a fast analyte diffusion because of an adequate molecular accommodation, which also preserved the penicillinase activity. This work therefore demonstrates the feasibility of employing LB films composed of lipids, CNTs, and enzymes as EIS devices for biosensing applications.
Algal polysaccharides (extracellular polysaccharides) and carbon nanotubes (CNTs) were adsorbed on dioctadecyldimethylammonium bromide Langmuir monolayers to serve as a matrix for the incorporation of urease. The physicochemical properties of the supramolecular system as a monolayer at the air–water interface were investigated by surface pressure–area isotherms, surface potential–area isotherms, interfacial shear rheology, vibrational spectroscopy, and Brewster angle microscopy. The floating monolayers were transferred to hydrophilic solid supports, quartz, mica, or capacitive electrolyte–insulator–semiconductor (EIS) devices, through the Langmuir–Blodgett (LB) technique, forming mixed films, which were investigated by quartz crystal microbalance, fluorescence spectroscopy, and field emission gun scanning electron microscopy. The enzyme activity was studied with UV–vis spectroscopy, and the feasibility of the thin film as a urea sensor was essayed in an EIS sensor device. The presence of CNT in the enzyme–lipid LB film not only tuned the catalytic activity of urease but also helped to conserve its enzyme activity. Viability as a urease sensor was demonstrated with capacitance–voltage and constant capacitance measurements, exhibiting regular and distinctive output signals over all concentrations used in this work. These results are related to the synergism between the compounds on the active layer, leading to a surface morphology that allowed fast analyte diffusion owing to an adequate molecular accommodation, which also preserved the urease activity. This work demonstrates the feasibility of employing LB films composed of lipids, CNT, algal polysaccharides, and enzymes as EIS devices for biosensing applications.