Sabato D'Auria

Enzyme fluorescence as a sensing tool: new perspectives in biotechnology

The technology for fluorescence protein-sensing is advancing rapidly owing to the continued introduction of new concepts, new fluorophores, and proteins engineered for sensing-specific analytes. Concerns about the reversibility and selectivity of engineered proteins are being addressed by developing biosensors that are based on the utilisation of coenzyme-depleted enzymes. Such biomolecules do not consume the substrate and can exhibit conformational changes upon the binding of the analyte, which can be easily detected as fluorescence change. In addition, concerns about the stability of biosensors can be overcome by using thermostable enzymes isolated from thermophilic microorganisms. Finally, the development of new techniques such as polarization-based sensing, anisotropy-based sensing and lifetime-based sensing, all of which can be accomplished with light-emitting diodes as the light source, is prompting the design of a new class of specific and stable biosensors, as has occurred with blood glucose measurement. These biosensors represent a valid alternative to the conventional clinical chemistry diagnostics.

Detection of odorant molecules via surface acoustic wave biosensor array based on odorant-binding proteins

In this paper, we present an array of biosensors for vapour phase detection of odorant molecules based on surface acoustic wave (SAW) resonators coated with odorant-binding proteins (OBPs). For the first time, the sensing capabilities of three different OBPs, as sensitive layers for SAW devices, are studied and compared. The SAW biosensor array is composed of three SAW devices coated by the droplet method with the wild-type OBP from cow (wtbOBP), a double mutant of the OBP from cow (dmbOBP) and the wild-type OBP from pig (wtpOBP). An uncoated device is used to compensate the variations of the environmental parameters. The SAW devices consist of two-port resonators fabricated on quartz (ST-cut, x propagation) with electrodes made of aluminium covered with a thin gold film (2 nm thick). The obtained surface densities of OBP layers are between 1.18×10(-6) kg/m(2) and 2.31×10(-6) kg/m(2) and were calculated measuring the resonant frequency shift of the SAW devices after the coating. The SAW biosensor array was tested in nitrogen upon exposure to vapours of R-(-)-1-octen-3-ol (octenol), in the range of concentration between 13 and 61 ppm, and R-(-)-carvone (carvone), in the range between 9 and 80 ppm. The highest sensitivity for detection of octenol (25.9 Hz/ppm) was obtained using the wtpOBP-based SAW biosensor, while the highest sensitivity for detection of carvone (9.2 Hz/ppm) was obtained using the dmbOBP-based SAW biosensor.

Proteins from extremophiles as stable tools for advanced biotechnological applications of high social interest

Extremophiles are micro-organisms adapted to survive in ecological niches defined as ‘extreme’ for humans and characterized by the presence of adverse environmental conditions, such as high or low temperatures, extreme values of pH, high salt concentrations or high pressure. Biomolecules isolated from extremophiles possess extraordinary properties and, in particular, proteins isolated from extremophiles represent unique biomolecules that function under severe conditions, comparable to those prevailing in various industrial processes. In this article, we will review some examples of recent applications of thermophilic proteins for the development of a new class of fluorescence non-consuming substrate biosensors for monitoring the levels of two analytes of high social interest, such as glucose and sodium.

Microbial carbohydrate esterases in cold adapted environments

Psychrophiles produce cold-evolved enzymes that display a high catalytic efficiency, associated with a low thermal stability. In recent years, these enzymes have attracted the attention of scientists because of their peculiar properties that render them particularly useful in investigating the relationship existing between enzyme stability and flexibility on one hand, and enzyme activity on the other hand. Among these enzymes, the esterases, and particularly the feruloyl esterases, have potential uses over a broad range of applications in the agro-food industries. In recent years, the number of microbial feruloyl esterase activities has increased in the growing genome databases. Based on substrate utilization data and supported by primary sequence identity, four subclasses of esterase have been characterized so far. Up to the present, ten genomes from psychrophilic bacteria have been completely sequenced and additional fourteen genomes are under investigation. From the bacteria strains whose genome has been completely sequenced, we analyzed the presence of esterase genes, both the putative genes and the determined experimentally genes, and performed a ClustalW analysis for feruloyl esterases. Major details will be presented for the ORF PSHAa1385 from P. haloplanktis TAC125 that recently has been studied in our research group. In addition, the potential biotechnology applications of this class of enzymes will be discussed.

Structure-function studies on β-glycosidase from Sulfolobus solfataricus. Molecular bases of thermostability

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a thermostable tetrameric protein with a molecular mass of 240 kDa which is stable in the presence of detergents and has a maximal activity above 95 degrees C. An understanding of the structure-function relationship of the enzyme under different chemical-physical conditions is of fundamental importance for both theoretical and application purposes. In this paper we report the effect of basic pH values on the structural stability of this enzyme. The structure of the enzyme was studied at pH 10 and in the temperature range 25-97.5 degrees C using circular dichroism, Fourier-transform infrared and fluorescence spectroscopy. The spectroscopic data indicated that the enzyme stability was strongly affected by pH 10 suggesting that the destabilization of the protein structure is correlated with the perturbation of ionic interactions present in the native protein at neutral pHs. These experiments give support to the observation derived from the 3D-structure, that large ion pair networks on the surface stabilize Sulfolobus solfataricus beta-glycosidase.

Patchy expression of lactase protein in adult rabbit and rat intestine

Enzymatic and immunohistological analyses of lactase were performed at different stages of development and within different regions of the small intestine of the rabbit and rat. As previously reported, there seems to be a sharp decline of lactase activity on weaning but variable and higher levels of activity are seen in adult animals. Two monoclonal antibodies to rat lactase were available to study the protein in rats. Four monoclonal antibodies to human lactase were shown to cross-react with rabbit lactase and used for the rabbit studies. Immunohistological analysis of small intestine of adult rabbits and rats showed residual lactase protein within the enterocytes throughout the small intestine. In the middle of the small intestine (lower jejunum, upper ileum), uniform staining of the brush border was observed. In the proximal and distal regions, a patchy pattern of staining was observed. This pattern, which resembles that observed in adult hypolactasic humans, indicates an underlying heterogeneity of enterocyte differentiation.

The Esterase From the Thermophilic Eubacterium Bacillus acidocaldarius: Structural-Functional Relationship and Comparison With the Esterase From the Hyperthermophilic Archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a “B‐type” carboxylesterase, displays the maximal activity at 65°C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT‐IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency‐domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in α‐helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent (2H2O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three‐exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473–481.

Myoglobin as a New Fluorescence Probe to Sense H2S

A new, fast, simple and cost-effective sensing device for monitoring H(2)S has been developed. Proof-of-principle results showing that a commercial and cheap Myoglobin (Mb) can be successfully used as a biological probe for a fluorescence biosensor for H(2)S detection are reported. The two different commercial labels Cy3 and Atto620 were selected for this study. A high selectivity for detecting H(2)S against other thiols was found. The applicability of the proposed sensing system was successfully explored not only in solution but also when applied in the form of a solid state device.

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein.

Despite the fact that the porcine odorant‐binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 Å) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (λmax = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (λmax = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat‐induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I320/I365) upon excitation at λex = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85°C, and the calculated midpoint of transition was centered at 70°C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature‐induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature. Proteins 2008.

Binding of glutamine to glutamine-binding protein from Escherichia coli induces changes in protein structure and increases protein stability

Glutamine‐binding protein (GlnBP) from Escherichia coli is a monomeric protein localized in the periplasmic space of the bacterium. It is responsible for the first step in the active transport of L‐glutamine across the cytoplasmic membrane. The protein consists of two similar globular domains linked by two peptide hinges, and X‐ray crystallographic data indicate that the two domains undergo large movements upon ligand binding. Fourier transform infrared spectroscopy (FTIR) was used to analyze the structure and thermal stability of the protein in detail. The data indicate that glutamine binding induces small changes in the secondary structure of the protein and that it renders the structure more thermostable and less flexible. Detailed analyses of IR spectra show a lower thermal sensitivity of α‐helices than β‐sheets in the protein both in the absence and in the presence of glutamine. Generalized two‐dimensional (2D) analyses of IR spectra reveal the same sequence of unfolding events in the protein in the absence and in the presence of glutamine, indicating that the amino acid does not affect the unfolding pathway of the protein. The data give new insight into the structural characteristics of GlnBP that are useful for both basic knowledge and biotechnological applications. Proteins 2005.