Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries

Background

The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.

Results

Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.

Conclusions

The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.

     

Lutein, zeaxanthin and astaxanthin protect against DNA damage in SK-N-SH human neuroblastoma cells induced by reactive nitrogen species

The purpose of this study was to evaluate the ability of the predominant carotenoids (lutein and zeaxanthin) of the macular pigment of the human retina, to protect SK-N-SH human neuroblastoma cells against DNA damage induced by different RNOS donors. Although astaxanthin has never been isolated from the human eye, it was included in this study because its structure is very close to that of lutein and zeaxanthin and because it affords protection from UV-light. DNA damage was induced by GSNO-MEE, a nitric oxide donor, by Na(2)N(2)O(3), a nitroxyl anion donor and by SIN-1, a peroxynitrite-generating agent. DNA damage was assessed using the comet assay, a rapid and sensitive single cell gel electrophoresis technique able to detect primary DNA damage in individual cells. The tail moment parameter was used as an index of DNA damage. The values of tail moment increased in all the samples incubated with the RNOS donors, indicating DNA impairment. Data obtained show that the ability of zeaxanthin, lutein, and astaxanthin to reduce the DNA damage depends on the type of RNOS donor and the carotenoid concentration used. All the carotenoids studied were capable of protecting against DNA damage in neuroblastoma cells when the cells were exposed to GSNO-MEE. However, a different behaviour was present when the other two RNOS donors were used. The presence of a carotenoid alone (without an RNOS donor) did not cause DNA damage. Spectrophotometric studies showed that the order with which tested carotenoids reacted with RNOS was not always in agreement with the DNA protection results. The data from this study provides additional information on the activities of the macular pigment carotenoids of the human retina.     

Antiproliferative and antiinflammatory coxib–combretastatin hybrids suppress cell cycle progression and induce apoptosis of MCF7 breast cancer cells

In our study, some newly synthesized aryl-substituted pyrazole derivatives mimicking cis-diphenylethylene scaffold of two apoptotic inducing agents celecoxib and combretastatin A-4 were found to have strong antiproliferative as well as antiinflammatory activities. Among these coxib–combretastatin hybrids, two lead compounds 8 and 6c simultaneously inhibited prostaglandin E2 (PGE2) production in LPS-activated murine macrophage RAW 264.7 cells and suppressed cell cycle progression of MCF7 cells at G2/M or G0/G1 phases, but only compound 8 induced apoptosis via caspase-3 activation. Both the lead compounds showed good docking energies with both protein targets COX-2 and tubulin in the molecule interaction modeling. The cis-diphenylethylene scaffold of celecoxib or combretastatin A-4 as well as functional groups such as the ethyl ester group and the sulfonamide could be considered as potential key features for the dual activity of studied compounds meanwhile the trimethoxybenzene remained the crucial characterization of the newly derived compounds of combretastatins.

     

Ligand effects on the structures and magnetic properties of tricyanomethanide-containing copper(II) complexes

The preparation, crystal structure and magnetic properties of four heteroleptic copper(II) complexes with the tricyanomethanide (tcm(-)) and the heterocyclic nitrogen donors 3,6-bis(2-pyridyl)pyridazine (dppn), 2,5-bis(2-pyridyl)pyrazine (2,5-dpp), 2,3-bis(2-pyridyl)pyrazine (2,3-dpp) and 2,3-bis(2-pyridyl)quinoxaline (2,3-dpq) are reported, {[Cu(2)(dppn)(OH)(tcm)(2)] x tcm}(n) (1), {[Cu(2,5-dpp)(tcm)] x tcm}(n) (2), {[Cu(2)(2,3-dpp)(2)(tcm)(3)(H(2)O)(0.5)] x tcm x 0.5H(2)O}(n) (3) and [Cu(2,3-dpq)(tcm)(2)](n) (4). 1 has a ladder-like structure with single mu-1,5-tcm ligands forming the sides and a bis-bidentate dppn and a single mu-hydroxo providing the rung. Each copper atom in 1 exhibits a distorted square pyramidal CuN(4)O surrounding: the basal plane is built by the hydroxo-oxygen, a nitrile-nitrogen atom from a tcm group and one pyrazine and a pyridyl nitrogen atoms from the dppn whereas the apical position is filled by a nitrile-nitrogen atom from a symmetry-related tcm ligand. The structures of 2-4 consists of zig-zag (2 and 3)/linear (4) chains of copper(II) ions which are bridged by either bis-bidentate 2,5-dpp (2) and 2,3-dpp (3) molecules or single mu-1,5-tcm (4) groups. The copper atoms in 2 and 4 are five coordinated with distorted trigonal bipyramidal (2) and square pyramidal (4) CuN(5) surroundings. The axial positions in 2 are occupied by two pyridyl-nitrogen atoms from two 2,5-dpp ligands whereas the trigonal plane is built by a nitrile-nitrogen from a terminally bound tcm group and two pyrazine nitrogen atoms from two 2,5-dpp molecules. The basal plane in 4 is defined by a pyridyl and a pyrazine nitrogen atoms from the bidentate 2,3-dpq ligand and two nitrile nitrogen atoms from two tcm groups (one terminal and the other bridging) whereas the apical position is filled by a nitrile nitrogen from another tcm ligand. The crystallographically independent copper atoms in 3 [Cu(1) and Cu(2)] exhibit elongated octahedral geometries being defined by four nitrogen atoms from two 2,3-dpp groups [Cu(1) and Cu(2)] either two terminally bound tcm ligands [Cu(1)] or a water molecule and a monodentate tcm ligand [Cu(2)] in cis positions. Magnetic susceptibility measurements for 1-4 in the temperature range 1.9-295 K reveal the occurrence of strong [J ca.-1000 cm(-1) (1); H = -JS(A) x S(B)] and weak [J = -0.13 (2), -0.67 (3) and -0.18 cm(-1) (4); H = -J Sigma(I)S(i) x S(i+1)] antiferromagnetic interactions in agreement with the different nature of the exchange pathways involved, diazine and single mu-hydroxo (1) and the extended 2,5-dpp (2), 2,3-dpp (3) and single mu-1,5-tcm (4) bridges with copper-copper separations of 3.363(8) (1), 7.111(1) (2), 6.823(1) and 7.056(1) (3) and 7.446(1) A (4).     

Analytical method for the determination of mycotoxins in indoor/outdoor airborne particulate matter by HPLC-MS-MS

An effective analytical method for the screening of mycotoxins, in indoor/outdoor airborne particulate matter, was developed and method performance data are presented. Mycotoxins are natural compounds produced, in particular conditions, as secondary metabolites by filamentous fungi and moulds, and, after their production, they can be transported far from their source. To simulate real samples, an urban dust (reference material 1649a) free from mycotoxins was used as matrix and spiked by the most common mycotoxins, chosen on the basis of studies carried out previously in other real matrices: deoxynivalenol, aflatoxin B1, ochratoxin A, T-2 toxin, zearalenone and sterigmatocystin. The analytical method was optimised and structured in four successive steps: (1) accelerated solvent extraction of the (spiked) analytes from matrix, (2) solid-phase purification (SPE) of the previous extract, (3) pre-concentration of the eluates from SPE and (4) analysis of the concentrated eluates by high performance liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode. After a proper sampling campaign, the method was applied to real indoor and outdoor particulate matter samples, where the clean-up step showed to be very effective and fundamental to avoid misleading analytical results.     

NMR depth profiles as a non-invasive analytical tool to probe the penetration depth of hydrophobic treatments and inhomogeneities in treated porous stones

Hydrophobic treatment is one of the most important interventions usually carried out in the conservation of stone artifacts and monuments. The analytical study reported in this paper was aimed at answering general questions such as the penetration depth of a hydrophobic treatment into a porous material, its capability to impair the water absorption, how the presence of a treatment may change the open porosity available to the water, and how a treatment may affect the diffusion of water inside a porous structure. Also, inhomogeneities in treated stones due to sharp variations of the amount of the absorbed product in the porous material were evidenced and scaled. The results of this fully non-invasive analytical study were rationalized in terms of new parameters obtained by a suitable process of nuclear magnetic resonance data. These analytical parameters reported here for the first time, namely the hydrophobic efficiency, the penetration depth, and angles describing changes in slope in depth profiles, gave important information in assessing the performance of a treatment.     

Unilateral NMR, 13C CPMAS NMR spectroscopy and micro-analytical techniques for studying the materials and state of conservation of an ancient Egyptian wooden sarcophagus

A multi-technique approach was employed to study a decorated Egyptian wooden sarcophagus (XXV–XXVI dynasty, Third Intermediate Period), belonging to the Museo del Vicino Oriente of the Sapienza University of Rome. Portable non-invasive unilateral NMR was applied to evaluate the conservation state of the sarcophagus. Moreover, using unilateral NMR, a non-invasive analytical protocol was established to detect the presence of organic substances on the surface and/or embedded in the wooden matrix. This protocol allowed for an educated sampling campaign aimed at further investigating the state of degradation of the wood and the presence of organic substances by ¹³C cross polarization magic angle spinning (CPMAS) NMR spectroscopy. The composition of the painted layer was analysed by optical microscopy (OM), scanning electron microscopy–energy dispersive spectroscopy (SEM–EDS), Raman and surface enhanced (resonance) Raman spectroscopy (SERS/SERRS), infrared and GC–MS techniques, evidencing original components such as clay minerals, Egyptian green, indigo, natural gums, and also highlighting restoration pigments and alteration compounds. The identification of the wood, of great value for the reconstruction of the history of the artwork, was achieved by means of optical microscopy.     

Potts q-color field theory and scaling random cluster model

We study structural properties of the q-color Potts field theory which, for real values of q, describes the scaling limit of the random cluster model. We show that the number of independent n-point Potts spin correlators coincides with that of independent n-point cluster connectivities and is given by generalized Bell numbers. Only a subset of these spin correlators enters the determination of the Potts magnetic properties for q integer. The structure of the operator product expansion of the spin fields for generic q is also identified. For the two-dimensional case, we analyze the duality relation between spin and kink field correlators, both for the bulk and boundary cases, obtaining in particular a sum rule for the kink-kink elastic scattering amplitudes.     

First magnetostructural study on a heterodinuclear 2,2'-bipyrimidine-bridged complex

The use of the [ReCl(4)(bpym)] precursor as a ligand toward the fully solvated nickel(II) metal ion affords the first example of a 2,2'-bipyrimidine-bridged Re(IV)-Ni(II) complex, [ReCl(4)(μ-bpym)NiBr(2)(H(2)O)(2)] (1), whose intramolecular ferromagnetic coupling has been substantiated from both experimental and theoretical studies.