Antonietta Parracino

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.

Nanostructured silicon-based biosensors for the selective identification of analytes of social interest

Small analytes such as glucose, L-glutamine (Gln), and ammonium nitrate are detected by means of optical biosensors based on a very common nanostructured material, porous silicon (PSi). Specific recognition elements, such as protein receptors and enzymes, were immobilized on hydrogenated PSi wafers and used as probes in optical sensing systems. The binding events were optically transduced as wavelength shifts of the porous silicon reflectivity spectrum or were monitored via changes of the fluorescence emission. The biosensors described in this article suggest a general approach for the development of new sensing systems for a wide range of analytes of high social interest.

Crystal structure of an S‐formylglutathione hydrolase from Pseudoalteromonas haloplanktis TAC125

S-formylglutathione hydrolases (FGHs) constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes, catalyzing the hydrolysis of S-formylglutathione to formic acid and glutathione. While a large number of functional studies have been reported on these enzymes, few structural studies have so far been carried out. In this article we report on the functional and structural characterization of PhEst, a FGH isolated from the psychrophilic bacterium Pseudoalteromonas haloplanktis. According to our functional studies, this enzyme is able to efficiently hydrolyze several thioester substrates with very small acyl moieties. By contrast, the enzyme shows no activity toward substrates with bulky acyl groups. These data are in line with structural studies which highlight for this enzyme a very narrow acyl-binding pocket in a typical alpha/beta-hydrolase fold. PhEst represents the first cold-adapted FGH structurally characterized to date; comparison with its mesophilic counterparts of known three-dimensional structure allowed to obtain useful insights into molecular determinants responsible for the ability of this psychrophilic enzyme to work at low temperature.

The Odorant-Binding Protein from Canis familiaris: Purification, Characterization and New Perspectives in Biohazard Assessment

In this report we show the purification to homogeneity and a partial characterization of a new odorant-binding protein from Canis familiar (CfOBP) nasal mucosa. In addition, we report preliminary data on the utilization of CfOBP as a probe for the development of a refractive index-based biosensor.

Glutamine-binding protein from Escherichia coli specifically binds a wheat gliadin peptide. 2. Resonance energy transfer studies suggest a new sensing approach for an easy detection of wheat gliadin.

In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutamine-binding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GlnBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food.

Fluorescence Biosensors for Continuously Monitoring the Blood Glucose Level of Diabetic Patients

Diabetes mellitus is increasing rapidly and will double in the next 15 years.1,2 In addition, diabetic patients have a mortality excess for cardiovascular disease up to 2.5-4 times more than non diabetic population.3,4 In the last years became evident a cluster of cardiovascular risk factors like hypertension, central obesity, dyslipidemia with low HDL-cholesterol and high triglycerides, impaired fibrinolysis, hypercoagulation and endothelial dysfunction that has been called metabolic syndrome.5 The underlying defect that shares all these alterations is insulin resistance with compensatory hyperinsulinemia that is associated with increased cardiovascular events and mortality.6,7 Patients with diabetes and/or metabolic syndrome must be treated aggressively about every risk factors to minimize the cardiovascular events. Hyperglycemia is clearly related to microvascular complication of diabetes: retinopathy, nephropathy and neuropathy, while for macrovascular complication other coexisting risk factors are important. Many studies have demonstrated that an intensive treatment of diabetes reduces the macro and microvascular complications and the best results are obtained when every risk factor is aggressively treated.8–10