Purification and characterization of a melanin biodegradation enzyme from Geotrichum sp.

OBJECTIVE: Melanin is a black or brown phenolic polymer present mainly in skin and hair. Although melanin can be degraded by some microbial species, the melanin degradation capacity of Geotrichum sp. is unknown. The aim of this study was to characterize a melanin biodegradation enzyme from Geotrichum sp. METHODS: In this study, we assessed the melanin degradation activity of Geotrichum sp. in comparison with the major melanin-degrading enzymes, manganese-dependent peroxidase (MnP), manganese-independent peroxidase, lignin peroxidase and laccase. Furthermore, the effect of several carbohydrates on melanin degradation by Geotrichum sp. was determined. The MnP enzyme was purified using ammonium sulphate precipitation and Sephadex G-200 column chromatography, and then the conditions for optimal enzymatic activity were determined by adjusting the pH, temperature and Tween-80 concentration. RESULTS: Compared with extracellular ligninolytic enzymes of Geotrichum sp., MnP had the highest ligninolytic enzyme activity; and the highest enzymatic activity was observed in the presence of glucose. The final purified MnP enzyme exhibited 6 U mL-1 activity and had a molecular weight of 54.2 kDa. The enzymatic activity was highest at pH 4.5 and 25-35°C in the absence of Tween-80. CONCLUSION: These results indicate the potential of MnP purified from Geotrichum sp. as a skin-lightening agent in the cosmetic industry.

Allochthonous bioaugmentation in ex situ treatment of crude oil-polluted sediments in the presence of an effective degrading indigenous microbiome.

Oil-polluted sediment bioremediation depends on both physicochemical and biological parameters, but the effect of the latter cannot be evaluated without the optimization of the former. We aimed in optimizing the physicochemical parameters related to biodegradation by applying an ex-situ landfarming set-up combined with biostimulation to oil-polluted sediment, in order to determine the added effect of bioaugmentation by four allochthonous oil-degrading bacterial consortia in relation to the degradation efficiency of the indigenous community. We monitored hydrocarbon degradation, sediment ecotoxicity and hydrolytic activity, bacterial population sizes and bacterial community dynamics, characterizing the dominant taxa through time and at each treatment. We observed no significant differences in total degradation, but increased ecotoxicity between the different treatments receiving both biostimulation and bioaugmentation and the biostimulated-only control. Moreover, the added allochthonous bacteria quickly perished and were rarely detected, their addition inducing minimal shifts in community structure although it altered the distribution of the residual hydrocarbons in two treatments. Therefore, we concluded that biodegradation was mostly performed by the autochthonous populations while bioaugmentation, in contrast to biostimulation, did not enhance the remediation process. Our results indicate that when environmental conditions are optimized, the indigenous microbiome at a polluted site will likely outperform any allochthonous consortium.

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor.

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.

Alkamides from Artemisia dracunculus.

From the aerial parts of Artemisia dracunculus, one known alkamide, pellitorine, two new alkamides neopellitorine A and neopellitorine B, and one known coumarin herniarine were isolated. Structures were elucidated by means of UV, IR, MS, 1H and 13C NMR. These compounds showed insecticidal activity against Sitophilus oryzae and Rhyzopertha dominica at 200 microg/ml concentrations.

Volatilization of mercury by immobilized bacteria (Klebsiella pneumoniae) in different support by using fluidized bed bioreactor.

Klebsiella pneumoniae, a mercury-resistant bacterial strain able to reduce ionic mercury to metallic mercury, was isolated from wastewater of Casablanca. This strain exhibits high minimal inhibition concentrations for heavy metals such as mercury 2400 microM, lead 8000 microM, silver 2400 microM, and cadmium 1000 microM. This bacterium was immobilized in alginate, polyacrylamide, vermiculite, and cooper beech and was used for removing mercury from a synthetic water polluted by mercury by using a fluidized bead bioreactor. Immobilized bacterial cells of Klebsiella pneumoniae could effectively volatilize mercury and detoxify mercury compounds. Moreover, the efficiency of mercury volatilization was much greater than with the native cells. The highest cleanup and volatilization rates were obtained when Klebsiella pneumoniae was entrapped in alginate beads, with a cleanup rate of 100% and a volatilization rate of 89%. Immobilized cells in alginate continuously volatilized mercury even after 10 days without loss of activity.

Study of paralytic shellfish poisoning toxin profile in shellfish from the Mediterranean shore of Morocco.

Since 1992, a monitoring program for bivalve molluscs contaminated by algal toxins was established at different stations along the Mediterranean Moroccan shores. The monitored stations were tested every 2 weeks. The presence of toxicity was determined using the mouse bioassay method. Toxin profile was carried out by HPLC/FD in selected contaminated tissues. According to the outcomes of this surveillance from 1994 to 1999, reliable information on toxicity of shellfish was obtained. They indicate that PSP is a recurrent toxicity in molluscs along the Mediterranean shore of Morocco. It has been noted a difference of PSP accumulation among individual shellfish. The cockle (Achanthocardia tuberculatum) presents toxicity throughout the year, while other specimens from the same area such as clam (Callista chione), warty venus (Venus gallina) and marine beans (Donax trunculus) accumulate it seasonally from January to April, after which they depurate the toxin. Moreover, the study of toxin profiles among individual shellfish was undertaken. It was found that shellfish presented a complex profile pointing to contamination by Gymnodinium catenatum.

Waste water bacterial isolates resistant to heavy metals and antibiotics.

Sewage water of Casablanca, an industrial city in Morocco, was studied for microorganisms resistant to heavy metals. Isolates were purified and collected on agar slants to be screened for resistance to heavy metals, including mercury in vitro. The strains that showed high resistance to heavy metals were also studied for their resistance to antibiotics and aromatic hydrocarbons. Results indicated that the strains most resistant to all tested products belonged to Ps. fluorescens, Ps. aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus sp. These strains exhibit high minimal inhibitory concentrations for heavy metals such as cadmium (2 mm) or mercury (1.2 mm). Growth of Ps. fluorescens and Klebsiella pneumoniae in the presence of heavy metals was also determined, and the growth curves indicated that mercury, copper, and zinc present a slight inhibitory action, while cadmium and silver could have a potent inhibitory action on growth compared with the controls. These studies also investigated growth in media containing aromatic compounds as the sole source of carbon. The results demonstrate that these strains could be good candidates for remediation of some heavy metals and aromatic compounds in heavily polluted sites.

Complexation and ionophoric properties of taxol and colchicine: complex formation and transport of sodium, potassium, magnesium and calcium ions across a liquid membrane.

We report the activities of taxol (an anticancer drug) and colchicine, which are inhibitors of microtubule organization, on the complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane, using a spectrophotometric procedure. Taxol, a diterpenoid compound, that has been demonstrated to possess a potent antitumour activity, is shown to extract Na+, K+, Mg2+ and Ca2+ ions from the aqueous solution to the organic phase with preference for Ca2+ ions. A kinetic study of the transport and complexation of Na+, K+, Mg2+ and Ca2+ ions through a liquid membrane revealed that the K+ ion is more rapidly transported and the Ca2+ ion is more rapidly complexed than other ions. However, colchicine, another alkaloid compound, extracted and transported only the divalent ions tested, Mg2+ and Ca2+. In both complexation and transport, the flux of the ions increases with the concentration of taxol or colchicine. Complexation and ionophoric properties of taxol and colchicine sheds new lights on therapeutic properties of these drugs. The treatment of disease states by the administration of these drugs to alter membrane permeability will prove to be a valuable therapeutic concept.

Okadaic acid and its interaction with sodium, potassium, magnesium and calcium ions: complex formation and transport across a liquid membrane.

Okadaic acid, a macrocyclic polyether compound, was shown to mediate the transfer of Na+, K+, Mg2+ and Ca2+ ions from aqueous solution to an organic phase, with a preference for Na+ ions. A kinetic study of the transport of these ions across a liquid membrane showed that the Na+ ion was more rapidly transported than the other ions and that the Na+ ion flux was dependent on the okadaic acid concentration.

Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14.

A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70,000. The isolated enzyme had a molecular weight of about 200,000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140,000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105,000 composed of two identical subunits of 52,000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.

Essential arginines in mercuric reductase isolated from Yersinia enterocolitica 138A14.

The mercuric reductase from Yersinia enterocolitica 138A14 was inactivated by the arginine modifying reagents 2,3-butanedione and phenylglyoxal. The inactivation by 2,3-butanedione exhibited second order kinetics with rate constant of 32 min-1 M-1. In the case of phenylglyoxal, biphasic kinetics were observed. The oxidized coenzyme (NADP+) prevented inactivation of the enzyme by the alpha-dicarbonyl reagents, whereas the reduced coenzyme (NADPH) enhanced the inactivation rate. The loss of enzyme activity was related to the incorporation of [2-14C] phenylglyoxal; when two arginines per subunit were modified the enzyme was completely inactivated.