Hydrolysis of polyethyleneterephthalate by p‐nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k_cat values decreased with increasing complexity of the substrate from 6 and 8 (s?1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s?1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Phytochemical analysis and in vitro evaluation of the biological activity against herpes simplex virus type 1 (HSV-1) of Cedrus libani A. Rich.

Cedrus libani are widely used as traditional medicine in Lebanon for treatment of different infection diseases. In the present study we reported the phytochemical composition analyzed by GC-MS of wood essential oil and cones and leaves ethanol extracts. The main components of wood essential oil were himachalol (22.50%), beta-himachalene (21.90%), and alpha-himachalene (10.50%). Leaves ethanol extract was characterized by a high content of germacrene d (29.40%). The same extract obtained from cones essentially contained alpha-pinene (51.0%) and beta-myrcene (13.0%). Moreover, we investigated extracts, essential oil, and identified compounds for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1). Cytotoxicity was evaluated by MTT assay in Vero cells. Cones and leaves ethanol extracts exhibited an interesting activity with IC50 of 0.50 and 0.66 mg/ml, respectively, at non-cytotoxic concentration. A comparable activity was found when essential oil was tested (IC50 of 0.44 mg/ml).     

Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus litoralis</Emphasis> for sugars monitoring

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection. The authors wish to dedicate this work to Prof. Ignacy Gryczynski, University of North Texas, TX, USA, for his outstanding contribution to the development of new sensing methodologies.     

KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactis

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.     

Exendin-4 Induces Cell Adhesion and Differentiation and Counteracts the Invasive Potential of Human Neuroblastoma Cells

Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R), thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i) the evaluation of neurite-like protrusions in 3D cell cultures, ii) the analysis of the expression of neuronal markers and iii) electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.     

Macrelmis pubescens (Grouvelle, 1889) – redescription of the species,designation of a lectotype and description of its larva (Coleoptera: Elmidae)

The adult of Macrelmis pubescens (Grouvelle, 1889) is redescribed, illustrated, a lectotype designated, and new locality information provided. The lectotype is housed in the Grouvelle’s collection in the Muséum national d’Histoire naturelle, Paris, France. The larva of M. pubescens is described, illustrated and compared with the known larvae of the genus. A diagnosis for Macrelmis Motschulsky, 1860 larvae is provided.