Eugenio Galano

Efficient immobilization of a fungal laccase and its exploitation in fruit juice clarification.

The clarification step represents, in fruit juices industries, a bottleneck process because residual phenols cause severe haze formation affecting juice quality and impairing customers acceptance. An enzymatic step can be efficiently integrated in the process, and use of immobilized enzymes entails an economical advantage. In this work, covalent immobilization of recombinant POXA1b laccase from Pleurotus ostreatus on epoxy activated poly(methacrylate) beads was optimized thanks to a Response Surface Methodologies approach. Through regression analysis the process was well fitted by a quadratic polynomial equation (R(2)=0.9367, adjusted R(2)=0.8226) under which laccase activity reached 2000 ± 100 Ug(-1) of beads, with an immobilization efficiency of 98%. The immobilized biocatalyst was characterized and then tested in fruit juice clarification reaching up to 45% phenol reduction, without affecting health-effective flavanones content. Furthermore, laccase treated juice displays an improved sensory profile, due to the reduction of vinyl guaiacol, a potent off-flavor possessing a peppery/spicy aroma.

A simple and reliable methodology to detect egg white in art samples

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th–16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.

Privileged Incorporation of Selenium as Selenocysteine in Lactobacillus reuteri Proteins Demonstrated by Selenium-specific Imaging and Proteomics

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS (78Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.

Comparison of the action of different proteases on virulence properties related to the staphylococcal surface

The purpose of this study was to evaluate the antimicrobial efficacy of five different proteases belonging to two different families on Staphylococcus aureus and Staphylococcus epidermidis strains.

A Biochemical and Cellular Approach to Explore the Antiproliferative and Prodifferentiative Activity of Aloe Arborescens Leaf Extract

Aloe arborescens Miller, belonging to the Aloe genus (Liliaceae family), is one of the main varieties of Aloe used worldwide. Although less characterized than the commonest Aloe vera, Aloe arborescens is known to be richer in beneficial phytotherapeutic, anticancer, and radio‐protective properties. It is commonly used as a pharmaceutical ingredient for its effect in burn treatment and ability to increase skin wound healing properties. However, very few studies have addressed the biological effects of Aloe at molecular level. The aim of the research is to provide evidences for the antiproliferative properties of Aloe arborescens crude leaf extract using an integrated proteomic and cellular biological approach. We analysed the composition of an Aloe arborescens leaf extract by gas chromatography‐mass spectrometry analysis. We found it rich in Aloe‐emodin, a hydroxylanthraquinone with known antitumoral activity and in several compounds with anti‐oxidant properties. Accordingly, we show that the Aloe extract has antiproliferative effects on several human transformed cell lines and exhibits prodifferentiative effects on both primary and immortalized human keratinocyte. Proteomic analysis of whole cell extracts revealed the presence of proteins with a strong antiproliferative and antimicrobial activity specifically induced in human keratinocytes by Aloe treatment supporting its application as a therapeutical agent. Copyright

A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions.

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.

Deglycosylation Step to Improve the Identification of Egg Proteins in Art Samples.

A deglycosylation step using Peptide-N-Glycosidase F (PNGaseF) has been introduced in a standard proteomic protocol to more confidently identify egg based binders. The ingenuity of introducing a PNGaseF digestion was aimed at removing the molecular hindrance, made up by the heavily glycosylated egg proteins, before the protease(s) hydrolysis. This novelty in the protocol resulted in obtaining a significant increase of proteolytic egg peptides thus improving the quality and reliability of egg identification in artwork samples. The protocol has been set up on paint replicas and successfully tested on two historical samples of different origin.

Qualitative screening in doping control by MALDI-TOF/TOF mass spectrometry: a proof-of-evidence.

The analysis of doping agents in biological fluids is of top significance in clinical and forensic toxicology. Herein we describe the study of a screening method for the detection of a mixture of drugs of potential abuse including cocaine and its metabolites. By using matrix-assisted laser desorption/ionization MALDI-TOF/TOF mass spectrometry. This screening procedure to detect the presence of different drugs, avoiding time consuming procedures could be useful in different fields of forensic analytical toxicology, including antidoping analysis.

GC/MS and proteomics to unravel the painting history of the lost Giant Buddhas of Bāmiyān (Afghanistan)

A chemical investigation of the organic paint binders of the Giant Buddhas of Bāmiyān was performed using an analytical approach based on mass spectrometry, combining traditional gas chromatography/mass spectrometry protocols with advanced proteomics methodologies. The research was carried out on a selection of rescued fragments. The data revealed the use of egg proteins as the paint binders of the original layers, in accordance with the traditional use of this proteinaceous medium in antiquity, spanning from the Mediterranean basin to the Far East, and already in the Bronze Age. Egg tempera was thus known to artists of the region in the first centuries AD, probably also due to the position of the Bāmiyān valley, which was connected to the Silk Road. Milk was found in the first historical overpaintings. A new proteomics approach was used, which was able to identify the source of the milk proteins present in the restoration layers, despite their age and degradation. In particular cow’s and goats milk were both found, in agreement with the documented presence of rich pastures in the Bāmiyān valley when the historical restorations were carried out. Investigating the materials of the Giant Buddhas not only enabled us to obtain isolated data on these invaluable works of art, which are now lost, but contributes to understanding the big “puzzle” of our past and the development of our culture, by implementing and supporting written sources, stylistic and anthropological studies with molecular data.