Influence of metals and metalloids on the composition and fluorescence quenching of the extracellular polymeric substances produced by the polymorphic fungus <i>Aureobasidium pullulans</i>

Aureobasidium pullulansis a ubiquitous and widely distributed fungus in the environment, and exhibits substantial tolerance against toxic metals. However, the interactions between metals and metalloids with the copious extracellular polymeric substances (EPS) produced byA. pullulansand possible relationships to tolerance are not well understood. In this study, it was found that mercury (Hg) and selenium (Se), as selenite, not only significantly inhibited growth ofA. pullulansbut also affected the composition of produced EPS. Lead (Pb) showed little influence on EPS yield or composition. The interactions of EPS fromA. pullulanswith the tested metals and metalloids depended on the specific element and their concentration. Fluorescence intensity measurements of the EPS showed that the presence of metal(loid)s stimulated the production of extracellular tryptophan-like and aromatic protein-like substances. Examination of fluorescence quenching and calculation of binding constants revealed that the fluorescence quenching process for Hg; arsenic (As), as arsenite; and Pb to EPS were mainly governed by static quenching which resulted in the formation of a stable non-fluorescent complexes between the EPS and metal(loid)s. Se showed no significant interaction with the EPS according to fluorescence quenching. These results provide further understanding of the interactions between metals and metalloids and EPS produced by fungi and their contribution to metal(loid) tolerance

Cupricyclins, Novel Redox-Active Metallopeptides Based on Conotoxins Scaffold

Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu2+ ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins

Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals

The expression of metal-binding proteins or peptides in microorganisms and plants in order to enhance heavy metal accumulation and/or tolerance has great potential. Several different peptides and proteins have been explored. This review focuses on cadmium (Cd) because of the significant importance of this metal and because of its global presence in many food materials

Tandem oligomeric expression of metallothionein enhance heavy metal tolerance and bioaccumulation in Escherichia coli.

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis and heavy metal detoxification. In our previous study, a novel full length MT cDNA was successfully cloned from the freshwater crab (Sinopotamon henanense). In the present study, tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR (SOEPCR) and expressed in Escherichia coli. The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins. The recombinant proteins were purified and their metal-binding abilities were further analyzed by the ultraviolet absorption spectral scan. Furthermore, the metal tolerance and bioaccumulation of E. coli cells expressing oligomeric MTs were determined. Results showed that the recombinant plasmids pET28a-SUMO-2MT and pET28a-SUMO-3MT were successfully constructed. SDS-PAGE analysis showed that the SUMO-2MT and SUMO-3MT were expressed mainly in the soluble forms. Oligomeric MTs expression significantly enhanced Cu, Cd or Zn tolerance and accumulation in E. coli in the order: SUMO-3MT˃SUMO-2MT˃SUMO-MT˃control. Cells harboring pET28a-SUMO -3MT exhibited the highest Cu, Cd or Zn bioaccumulation at 5.8-fold, 3.1-fold or 6.7-fold higher than that of the control cells. Our research could lay a foundation for large-scale preparation of MTs and provide a scientific basis for bioremediation of heavy metal pollution by oligomeric MTs

Examining the Effects of the Destroying Ammunition, Mines, and Explosive Devices on the Presence of Heavy Metals in Soil of Open Detonation Pit: Part 1-Pseudo-total Concentration

This paper presents the results of determining the pseudo-total concentration of five heavy metals in the soil on which the destruction of ammunition, mines, and explosive devices is carried out by the method of open detonation. In the analyzed area, the concentrations of cadmium, lead, nickel, copper, and zinc were determined, while from the physical properties of the soil were determined the granulometric composition and the pH. The aim of the study is to determine the origin and total load on heavy metals and, based on that, to assess the dangers and impact of the site in terms of the soil pollution by heavy metals. In accordance with the regulations of Bosnia and Herzegovina, the results of the soil testing showed a significant load of copper (up to seven times) and cadmium (up to six times), and exceeding the allowed values for nickel and zinc in some places. Lead was the only metal whose concentration was within the maximum allowed and according to that the soil was classified as unpolluted. A sample of soil from the edge of the pit is the only sample in which all heavy metals, except Ni, were within the maximum allowable concentration. In regard to the concentration of the examined metals, the soil of the pit is classified as medium polluted from the aspect of copper, cadmium, and nickel and highly contaminated with zinc. The concentrations of copper and zinc in the examination area correspond to contaminated soil that represents ecological risk, which requires soil remediation