Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats.

The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5°C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65°C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16°C, was shown to contain halopiezophilic-like enzymes that are most active at 70°C and with denaturing temperatures of 71.4°C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.

Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp Rimicaris exoculata by using functional metagenomics.

The shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (≤52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (≤356 U mg(-1)) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities.

Three-dimensional structure of Saccharomyces invertase: role of a non-catalytic domain in oligomerization and substrate specificity.

Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic ß-propeller and ß-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a ß-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the ß-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition.

Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/ß hydrolase family.

Several members of the C-C MCP (meta-cleavage product) hydrolase family demonstrate an unusual ability to hydrolyse esters as well as the MCPs (including those from mono- and bi-cyclic aromatics). Although the molecular mechanisms responsible for such substrate promiscuity are starting to emerge, the full understanding of these complex enzymes is far from complete. In the present paper, we describe six distinct α/ß hydrolases identified through genomic approaches, four of which demonstrate the unprecedented characteristic of activity towards a broad spectrum of substrates, including p-nitrophenyl, halogenated, fatty acyl, aryl, glycerol, cinnamoyl and carbohydrate esters, lactones, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate and 2-hydroxy-6-oxohepta-2,4-dienoate. Using structural analysis and site-directed mutagenesis we have identified the three residues (Ser32, Val130 and Trp144) that determine the unusual substrate specificity of one of these proteins, CCSP0084. The results may open up new research avenues into comparative catalytic models, structural and mechanistic studies, and biotechnological applications of MCP hydrolases.

Crystallization and preliminary X-ray diffraction analysis of the invertase from Saccharomyces cerevisiae.

Saccharomyces cerevisiae invertase (ScInv) is an enzyme encoded by the SUC2 gene that releases ß-fructose from the nonreducing termini of various ß-D-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which presents a high degree of oligomerization, was crystallized by vapour-diffusion methods. The crystals belonged to space group P3(1)21, with unit-cell parameters a=268.6, b=268.6, c=224.4 Å. The crystals diffracted to 3.3 Šresolution and gave complete data sets using a synchrotron X-ray source.

Development of an Electrospun Patch Platform Technology for the Delivery of Carvedilol in the Oral Mucosa.

The work herein presented aims to develop and characterize carvedilol (CVD) releasable non-water-soluble monolayers and a multilayer patch made of ultrathin micron and submicron fibers for drug delivery into the sublingual mucosa. Firstly, the developed formulations containing CVD within different biopolymers (PDLA, PCL, and PHB) were characterized by scanning electron microscopy (SEM), attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), wide-angle X-ray scattering (WAXS), and for their in vitro drug release. SEM micrographs assessed the fiber morphology attained by adding carvedilol. ATR-FTIR spectra revealed good chemical compatibility between CVD and the tested biopolymers, whereas DSC and WAXS confirmed that CVD was in an amorphous state within the biopolymeric fibers. In vitro release studies showed enhanced CVD release kinetics from the electrospun biopolymer monolayers compared to the dissolution rate of the commercial form of the pure drug, except for the slow-releasing PDLA fibers. Finally, the selected CVD-loaded layer, i.e., electrospun PHB, was built into a three-layer patch to tackle mucosa adhesion and unidirectional release, while retaining the enhanced release kinetics. The patch design proposed here further demonstrates the potential of the electro-hydrodynamic processing technology to render unique mucoadhesive controlled delivery platforms for poorly water-soluble drugs.

Fructo-oligosaccharide synthesis by mutant versions of Saccharomyces cerevisiae invertase.

Efficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered the Saccharomyces cerevisiae invertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (ß-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lower substrate (sucrose) affinity produced FOS with longer half-lives. A mutation (P205V) adjacent to another conserved motif (EC) caused a 6-fold increment in 6-kestose yield. Docking studies with a Suc2 modeled structure defined a putative acceptor substrate binding subsite constituted by Trp 291 and Asn 228. Mutagenesis studies confirmed the implication of Asn 228 in directing the orientation of the sucrose molecule for the specific synthesis of ß(2,6) linkages.

Super-Repellent Paper Coated with Electrospun Biopolymers and Electrosprayed Silica of Interest in Food Packaging Applications.

In the current work, a super-repellent biopaper suitable for food contact applications was developed. To do this, three different kinds of biopolymers, namely polylactide (PLA), poly(ε-caprolactone) (PCL), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and hydrophobic silica microparticles (SiO2), were sequentially processed by electrohydrodynamic processing (EDHP). As a first step, the ultrathin biopolymer fibers were deposited onto a commercial food contact cellulose paper by electrospinning and, thereafter, the nanostructured silica was sequentially electrosprayed. The multilayer coated papers were annealed at different temperatures to promote adhesion between the layers and enhance the super-repellent properties. The developed coatings were characterized in terms of morphology, permeance to water vapor, adhesion, mechanical resistance, and contact and sliding angle. The resultant multilayer biopapers presented a hierarchical micro/nanostructured surface with an apparent water contact angle (WCA) higher than 155° and sliding angle (SA) lower than 10° for all the tested biopolymers used. Among the different multilayer approaches, it was observed that the paper/PHBV/SiO2 showed the best performance, in terms of water vapor permeance; resistance after the tape peeling-off test; and food super-repelling properties to water, yogurt, and custard. Overall, this study presents the successful generation of super-repellent biopapers coated with PLA, PCL, or PHBV along with hydrophobic silica microparticles and its effectiveness for easy emptying food packaging applications to reduce food waste.

Bioremediation of Southern Mediterranean oil polluted sites comes of age.

Mediterranean Sea is facing a very high risk of oil pollution due to the high number of oil extractive and refining sites along the basin coasts, and the intense maritime traffic of oil tankers. All the Mediterranean countries have adopted severe regulations for minimizing pollution events and bioremediation feasibility studies for the most urgent polluted sites are undergoing. However, the analysis of the scientific studies applying modern 'meta-omics' technologies that have been performed on marine oil pollution worldwide showed that the Southern Mediterranean side has been neglected by the international research. Most of the studies in the Mediterranean Sea have been done in polluted sites of the Northern side of the basin. Those of the Southern side are poorly studied, despite many of the Southern countries being major oil producers and exporters. The recently EU-funded research project ULIXES has as a major objective to increase the knowledge of the bioremediation potential of sites from the Southern Mediterranean countries. ULIXES is targeting four major polluted sites on the coastlines of Egypt, Jordan, Morocco and Tunisia, including seashore sands, lagoons, and oil refinery polluted sediments. The research is designed to unravel, categorize, catalogue, exploit and manage the diversity and ecology of microorganisms thriving in these polluted sites. Isolation of novel hydrocarbon degrading microbes and a series of state of the art 'meta-omics' technologies are the baseline tools for improving our knowledge on biodegradation capacities mediated by microbes under different environmental settings and for designing novel site-tailored bioremediation approaches. A network of twelve European and Southern Mediterranean partners is cooperating for plugging the existing gap of knowledge for the development of novel bioremediation processes targeting such poorly investigated polluted sites.

Fermentation of cellobiose to ethanol by industrial Saccharomyces strains carrying the ß-glucosidase gene (BGL1) from Saccharomycopsis fibuligera.

Constructs carrying the Saccharomycopsis fibuligera ß-glucosidase gene (BGL1) under the control of a constitutive actin or a galactose-inducible promoter were introduced into eleven Saccharomyces strains. In ten of these recombinant strains, BGL1 expression driven by the actin promoter was between 1.6- and 18-fold higher than that obtained with the galactose-inducible promoter. Strains carrying the actin promoter yielded ethanol concentrations from cellobiose of between 0.5% and 14%, depending on their ability to accumulate Bgl1 (between 30 and 250 mU/mL) but also on their genetic background. Comparative analysis of a S. cerevisiae strain and its corresponding petite version showed similar ethanol yields, despite a 3-fold lower ß-glucosidase production of the latter, suggesting that respiratory activity could be one of the factors influencing ethanol production when using carbon sources other than glucose. This study provides a selection of strains that may be good candidates as hosts for ethanol biosynthesis from cellulosic substrates.