Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremediation because of their improved thermal, chemical, and mechanical stability with respect to enzymes in solution. The B isozyme of catechol 1,2 dioxygenase from Acinetobacter radioresistens and its mutants of Leu69 and Ala72, designed for an increased reactivity toward the environmental pollutant chlorocatechols, were encapsulated using alkoxysilanes and alkyl alkoxysilanes as precursors in varying proportions. Encapsulation of the mutants in a hydrophobic tetramethoxysilane/dimethoxydimethylsilane-based matrix yielded a remarkable 10- to 12-fold enhancement in reactivity toward chlorocatechols. These gels also showed a fivefold increase in relative reactivity toward chlorocatechols with respect to the natural substrate catechol, thus compensating for their relatively low activity for these substrates in solution. The encapsulated enzyme, unlike the enzyme in solution, proved resilient in assays carried out in urban wastewater and bacteria-contaminated solutions mimicking environmentally relevant conditions. Overall, the combination of a structure-based rational design of enzyme mutants, and the selection of a suitable encapsulation material, proved to be a powerful approach for the production and optimization of a potential bioremediation device, with increased activity and resistance toward bacterial degradation.

SUMMARY In Bufo bufo larvae the action of STH and LTH on body weight, processes of metamorphosis, and the cytologic pattern of the hypophysis and thyroid glands were studied. Furthemore, biochemical investigations were carried on the liver glycogen content and some enzymes involved in its synthesis and breakdown. Generally, STH-treated larvae underwent a loss in body weight, which might be related to the acceleration of metamorphosis shown by them as compared to controls. A pattern of definite hyperfunction is exhibited by the thyroid; in the adenohypophysis thyrotrophs result much degranulated. A decrease in the liver glycogen content and a decline in the activity of glycogen-synthetase was caused by STH, while phosphorylase activity was unchanged. In LTH-treated larvae, a statistically significant increase in body weight higher than in controls was seen to occur. The unfolding of metamorphosis in the early stages, at least, resulted wholly similar to that of controls, although in the treated larvae a mo...

SUMMARY Historadiography was used to study tyrosinase activity in melanophores and the possible influence of MSH. 2G14-tyrosine was injected into Xenopus laevis larvae, under both normal and experimental conditions (namely, larvae with hypertrophy of the pituitary intermediate lobe and treated with methylthiouracil, and larvae treated with thiouracil only). A scanty tracer uptake was seen to occur in all animal groups. FISZPATRICK & KUKITA'S method for tyrosinase detection revelead that: 1. 2C14-tyrosine is incorporated into the melanophores of normal larvae at all developmental stages examined (stages 50–54); 2. there is a higher 2C14-tyrosine uptake by melanophores of larvae treated with thiouracil only, compared with those of operated and thiouraciltreated larvae. Biochemical investigations revealed an overall increase in melanin amount in the larvae with a hypertrophic pituitary intermediate lobe with respect to normal larvae. These results are briefly discussed.

Intradiol dioxygenase are iron-containing enzymes involved in the bacterial degradation of natural and xenobiotic aromatic compounds. The wild-type and mutants forms of catechol 1,2-dioxygenase Iso B from Acinetobacter radioresistens LMG S13 have been investigated in order to get an insight on the structure-function relationships within this system. 4K CW-EPR spectroscopy highlighted different oxygen binding properties of some mutants with respect to the wild-type enzyme, suggesting that a fine tuning of the substrate-binding determinants in the active site pocket may indirectly result in variations of the iron reactivity. A thermostability investigation by optical spectroscopy, that reports on the state of the metal center, showed that the structural stability is more influenced by the type rather than by the position of the mutation. Finally, the influence of pH and temperature on the catalytic activity was monitored and discussed in terms of perturbations induced on the tertiary contact network of the enzyme.

Selenium (Se), Se-cysteines and selenoproteins have received growing interest in the nutritional field as redox-balance modulating agents. The aim of this study was to establish the Se-concentrating and Se-metabolizing capabilities of the probiotic Lactobacillus reuteri Lb2 BM, for nutraceutical applications. A comparative proteomic approach was employed to study the bacteria grown in a control condition (MRS modified medium) and in a stimulated condition (4.38 mg/L of sodium selenite). The total protein extract was separated into two pI ranges: 4-7 and 6-11; the 25 identified proteins were divided into five functional classes: (i) Se metabolism; (ii) energy metabolism; (iii) stress/adhesion; (iv) cell shape and transport; (v) proteins involved in other functions. All the experimental results indicate that L. reuteri Lb2 BM is able to metabolize Se(IV), incorporating it into selenoproteins, through the action of a selenocysteine lyase, thus enhancing organic Se bioavailability. This involves endo-ergonic reactions balanced by an increase of substrate-level phosphorylation, chiefly through lactic fermentation. Nevertheless, when L. reuteri was grown on Se a certain degree of stress was observed, and this has to be taken into account for future applicative purposes. The proteomic approach has proven to be a powerful tool for the metabolic characterization of potential Seconcentrating probiotics.

In the present study, the high isoelectric point sub-proteome of Acinetobacter radioresistens S13 grown on aromatic compounds (benzoate or phenol) was analyzed and compared to the protein pattern, in the same pI range, of acetate-grown bacteria (control condition). Analyses concerned both soluble and membrane enriched proteomes and led to the identification of 25 proteins that were differentially expressed among the growth conditions considered: most of them were up-regulated in cells grown on aromatic compounds. Up to 17 identified proteins can be, more or less directly, related to the so called "envelope stress responses": these signal transduction pathways are activated when bacterial cells are exposed to stressing environments (e.g., heat, pH stress, organic solvents, osmotic stress) causing accumulation of misfolded/unfolded cell wall proteins into the periplasmic space. For, at least, five of these proteins (a DegP-like serine protease, a peptidyl-prolyl cis-trans isomerase, a phosphatidylserine decarboxylase, a pseudouridine synthase, and a TolB-like protein) a direct induction via either the σ(E) or the Cpx alternative signalling systems mediating envelope stress responses was previously demonstrated in Gram-negative bacteria. The proteins identified in this study include periplasmic proteases, chaperones, enzymes catalyzing peptydoglycan biogenesis, proteins involved in outer membrane integrity, cell surface properties and cellular redox homeostasis. The present study brings additional information to previous works on the acidic proteome of A. radioresistens S13, thus complementing and refining the metabolic picture of this bacterial strain during growth on aromatic compounds.