Enhancement of antibiotics antimicrobial activity due to the silver nanoparticles impact on the cell membrane.

The ability of microorganisms to generate resistance outcompetes with the generation of new and efficient antibiotics; therefore, it is critical to develop novel antibiotic agents and treatments to control bacterial infections. An alternative to this worldwide problem is the use of nanomaterials with antimicrobial properties. Silver nanoparticles (AgNPs) have been extensively studied due to their antimicrobial effect in different organisms. In this work, the synergistic antimicrobial effect of AgNPs and conventional antibiotics was assessed in Gram-positive and Gram-negative bacteria. AgNPs minimal inhibitory concentration was 10-12 µg mL-1 in all bacterial strains tested, regardless of their different susceptibility against antibiotics. Interestingly, a synergistic antimicrobial effect was observed when combining AgNPs and kanamycin according to the fractional inhibitory concentration index, FICI: <0.5), an additive effect by combining AgNPs and chloramphenicol (FICI: 0.5 to 1), whereas no effect was found with AgNPs and ß-lactam antibiotics combinations. Flow cytometry and TEM analysis showed that sublethal concentrations of AgNPs (6-7 µg mL-1) altered the bacterial membrane potential and caused ultrastructural damage, increasing the cell membrane permeability. No chemical interactions between AgNPs and antibiotics were detected. We propose an experimental supported mechanism of action by which combinatorial effect of antimicrobials drives synergy depending on their specific target, facilitated by membrane alterations generated by AgNPs. Our results provide a deeper understanding about the synergistic mechanism of AgNPs and antibiotics, aiming to combat antimicrobial infections efficiently, especially those by multi-drug resistant microorganisms, in order to mitigate the current crisis due to antibiotic resistance.

Synthesis and Complete Antimicrobial Characterization of CEOBACTER, an Ag-Based Nanocomposite.

The antimicrobial activity of silver nanoparticles (AgNPs) is currently used as an alternative disinfectant with diverse applications, ranging from decontamination of aquatic environments to disinfection of medical devices and instrumentation. However, incorporation of AgNPs to the environment causes collateral damage that should be avoided. In this work, a novel Ag-based nanocomposite (CEOBACTER) was successfully synthetized. It showed excellent antimicrobial properties without the spread of AgNPs into the environment. The complete CEOBACTER antimicrobial characterization protocol is presented herein. It is straightforward and reproducible and could be considered for the systematic characterization of antimicrobial nanomaterials. CEOBACTER showed minimal bactericidal concentration of 3 µg/ml, bactericidal action time of 2 hours and re-use capacity of at least five times against E. coli cultures. The bactericidal mechanism is the release of Ag ions. CEOBACTER displays potent bactericidal properties, long lifetime, high stability and re-use capacity, and it does not dissolve in the solution. These characteristics point to its potential use as a bactericidal agent for decontamination of aqueous environments.

Enhancement of Peroxidase Stability Against Oxidative Self-Inactivation by Co-immobilization with a Redox-Active Protein in Mesoporous Silicon and Silica Microparticles.

The study of the stability enhancement of a peroxidase immobilized onto mesoporous silicon/silica microparticles is presented. Peroxidases tend to get inactivated in the presence of hydrogen peroxide, their essential co-substrate, following an auto-inactivation mechanism. In order to minimize this inactivation, a second protein was co-immobilized to act as an electron acceptor and thus increase the stability against self-oxidation of peroxidase. Two heme proteins were immobilized into the microparticles: a fungal commercial peroxidase and cytochrome c from equine heart. Two types of biocatalysts were prepared: one with only covalently immobilized peroxidase (one-protein system) and another based on covalent co-immobilization of peroxidase and cytochrome c (two-protein system), both immobilized by using carbodiimide chemistry. The amount of immobilized protein was estimated spectrophotometrically, and the characterization of the biocatalyst support matrix was performed using Brunauer-Emmett-Teller (BET), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX), and Fourier transform infrared (FTIR) analyses. Stability studies show that co-immobilization with the two-protein system enhances the oxidative stability of peroxidase almost four times with respect to the one-protein system. Thermal stability analysis shows that the immobilization of peroxidase in derivatized porous silicon microparticles does not protect the protein from thermal denaturation, whereas biogenic silica microparticles confer significant thermal stabilization.

Effect of broccoli (Brassica oleracea) and its phytochemical sulforaphane in balanced diets on the detoxification enzymes levels of tilapia (Oreochromis niloticus) exposed to a carcinogenic and mutagenic pollutant.

Tilapia fish (Oreochromis niloticus) were fed with enriched diets containing broccoli and its phytochemical sulforaphane over 30 d. The levels of cytochrome P450, superoxide dismutase, catalase, lipid peroxidation and glutathione-S-transferase activities were measured. Basal value of cytochrome P450 activity was significantly increased as consequence of the broccoli and sulforaphane enriched diets, while no statistically significant changes were found on catalase and lipid peroxidation activities. After benzo(a)pyrene exposure, the cytochrome P450 activity increased to higher levels in the fish feed with broccoli and sulforaphane when compared with the control fish. Activities of antioxidant enzymes also varied but without significant difference with the control fish. Supported by the lower concentrations of BaP metabolites in bile from fish fed with broccoli or with sulforaphane enriched diets (indicating a better xenobiotic elimination) the cytochrome P450 induction could be considered beneficial for the detoxification because this transformation is the first step for PAH elimination by the phase II system. The protection of aquaculture organism against pollution effects by designing special diets able to modulate the enzymes involved in the phase-I and phase-II detoxification mechanism are discussed.

Chemical modification of turnip peroxidase with methoxypolyethylene glycol enhances activity and stability for phenol removal using the immobilized enzyme.

Peroxidase from turnip roots (TP) was isolated followed by modification with methoxypolyethylene glycol (MPEG). The catalytic activity of the modified TP (MTP) on ABTS increased 2.5 times after 80 min of reaction. MTP showed a KM similar value to that of TP, but a significantly greater kcat for ABTS oxidation, in aqueous buffer. Chemical modification produced an enhanced stability in organic solvents and increased thermal stability of about 4 times that of TP, in aqueous buffer at 70 degrees C. Circular dichroism showed that MPEG modification decreased TP alpha-helical structure from 26 to 16% and increased beta-turns from 26 to 34%, resulting in an enhanced conformational stability. The temperature at the midpoint of thermal denaturation (melting temperature) increased from 57 to 63 degrees C after modification. MTP was immobilized in alginate beads (IMTP) and tested for oxidative polymerization of concentrated phenolic synthetic solutions, achieving 17 effective contact cycles removing >65% phenols. IMTP may be useful for the development of an enzymatic process for wastewater effluent treatment.

Polyethylene glycol improves phenol removal by immobilized turnip peroxidase.

Purified peroxidase from turnip (Brassica napus L. var. esculenta D.C.) was immobilized by entrapment in spheres of calcium alginate and by covalent binding to Affi-Gel 10. Both immobilized Turnip peroxidase (TP) preparations were assayed for the detoxification of a synthetic phenolic solution and a real wastewater effluent from a local paints factory. The effectiveness of phenolic compounds (PC's) removal by oxidative polymerization was evaluated using batch and recycling processes, and in the presence and in the absence of polyethylene glycol (PEG). The presence of PEG enhances the operative TP stability. In addition, reaction times were reduced from 3h to 10 min, and more effective phenol removals were achieved when PEG was added. TP was able to perform 15 reaction cycles with a real industrial effluent showing PC's removals >90% PC's during the first 10 reaction cycles. High PC's removal efficiencies (>95%) were obtained using both immobilized preparations at PC's concentrations <1.2mM. Higher PC's concentrations decreased the removal efficiency to 90% with both preparations after the first reaction cycle, probably due to substrate inhibition. On the other hand, immobilized TP showed increased thermal stability when compared with free TP. A large-scale enzymatic process for industrial effluent treatment is expected to be developed with immobilized TP that could be stable enough to make the process economically feasible.

Role of phenanthrene in rhamnolipid production by P. putida in different media.

The role of phenanthrene in rhamnolipid production by P. putida in eight media with different culture conditions was investigated. Cultures using Fe2SO4.7H2O, KH2PO4, NH4Cl, yeast extract, glucose, and corn oil, with and without 200 mg l(-1) of phenanthrene, were evaluated under shaking for rhamnolipid production through a 2(7-4) fractional factorial experimental design. The biosurfactant production, decrease in the surface tension of the broth and the total cell growth in media without phenanthrene were affected significantly (p < 0.001) by yeast extract, glucose, corn oil and NH4Cl, and in media with phenanthrene by glucose and yeast extract. The non polar fraction of the biosurfactant in all media was composed of linoleic (C18:2), arachidic (C20:0) and behenic (C22:0) fatty acids. The medium with phenanthrene (200 mg l(-1)), Fe2SO4.7H2O (5 x 10(-4) g l(-1)), KH2PO4 (0.2 g l(-1)), glucose (50 g l(-1)), yeast extract (1 g l(-1)), corn oil (2% vol), and NH4Cl (1 g l(-1)), shaken at 150 rpm at 37 degrees C, and pH 7.0, presented the highest biosurfactant production. For this medium the surface tension decreased by 35.9 mN m(-1) in relation to the initial value, and only this medium showed an emulsion capacity of 20%. The polar fraction (Rhamnose) in media 1, 3, 7 and 8 with phenanthrene was c.a 100%, in contrast to those without phenanthrene where this fraction was undetectable.

Enhanced activity by poly(ethylene glycol) modification of Coriolopsis gallica laccase.

We are studying the enzymatic modification of polycyclic aromatic hydrocarbons (PAHs) by the laccase from Coriolopsis gallica UAMH 8260. The enzyme was produced during growth in a stirred tank reactor to 15 units ml(-1), among the highest levels described for a wild-type fungus; the enzyme was the major protein produced under these conditions. After purification, it exhibited characteristics typical of a white rot fungal laccase. Fifteen azo and phenolic compounds at 1 mM concentration were tested as mediators in the laccase oxidation of anthracene. Higher anthracene oxidation was obtained with the mediator combination of ABTS and HBT, showing a correlation between the oxidation rate and the mediator concentration. Reactions with substituted phenols and anilines, conventional laccase substrates, and PAHs were compared using the native laccase and enzyme preparations chemically modified with 5000 MW-poly(ethylene glycol). Chemically modified laccase oxidized a similar range of substituted phenols as the native enzyme but with a higher catalytic efficiency. The k(cat) increase by the chemical modification may be as great as 1300 times for syringaldazine oxidation. No effect was found of chemical modification on mediated PAH oxidation. Both unmodified and PEG-modified laccases increased PAH oxidation up to 1000 times in the presence of radical mediators. Thus, a change of the protein surface improves the mediator oxidation efficiency, but does not affect non-enzymatic PAH oxidation by oxidized mediators.

Enzymatic coupling of phenol vapors onto chitosan.

Phenols are important industrial chemicals, and because they can be volatile, also appear as air pollutants. We examined the potential of tyrosinase to react with the volatile phenol p-cresol. Three lines of evidence support the conclusion that volatile phenols react with tyrosinase and are coupled (i.e., chemisorbed) onto chitosan films. First, phenol-trapping studies indicated that p-cresol can be removed from vapors if the vapors are contacted with tyrosinase-coated chitosan films. Second, the ultraviolet absorbance of tyrosinase-coated chitosan films changes dramatically when they are contacted with cresol-containing vapors, whereas control films are unaffected by contacting with cresol vapors. Third, pressure measurements indicate that tyrosinase-coated chitosan films only react with cresol vapors if the oxygen cosubstrate is present. Additional studies demonstrate the potential of tyrosinase-coated chitosan films/membranes for the detection and removal of phenol vapors.

Combinatorial screening for enzyme-mediated coupling. Tyrosinase-catalyzed coupling to create protein--chitosan conjugates.

In nature, tyrosinase-generated o-quinones are commonly involved in processes that lead to functional biomaterials. These biomaterials are chemically complex and have been difficult to analyze. Furthermore, the cascade of reactions involving o-quinones is poorly understood, and it has been difficult to mimic ex vivo for materials processing. We report the use of a combinatorial approach to learn how tyrosinase and low molecular weight phenolic precursors can be used to generate biologically active protein-polysaccharide conjugates. Specifically, we screened various phenolic coupling precursors and various reaction conditions for the coupling of proteins onto the polysaccharide chitosan. Several natural phenols were identified as appropriate precursors for the coupling of polyhistidine tagged organophosphorus hydrolase (His-OPH) onto chitosan films. OPH activity was retained upon coupling and subsequent studies indicated that the histidine tag was not necessary for coupling. Using conditions identified for His-OPH coupling, we observed that various biologically active proteins (cytochrome c, OPH, and His-CAT) could be coupled onto chitosan films. The glycosylated protein horseradish peroxidase was not effectively coupled onto chitosan under the conditions studied. In all cases studied, we observed that coupling required a phenolic precursor, suggesting that tyrosinase is unable to couple by reaction with surface tyrosyl residues of the target protein. In conclusion, this study illustrates a combinatorial approach for the "discovery" of conditions to couple biologically active proteins onto chitosan through natural, quinone-based processes.

Biocatalytic chlorination of aromatic hydrocarbons by chloroperoxidase of Caldariomyces fumago.

Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min(-1) for naphthalene to 758 min(-1) for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.

Kinetic differences of purified laccases from six Pleurotus ostreatus strains.

AIMS: Enzyme kinetics of purified laccases from six different Pleurotus ostreatus strains were determined in the oxidation of syringaldazine, guaiacol and ABTS. METHODS AND RESULTS: Significant differences in the kinetic constants were found. Catalytic activity (kcat) ranged from 19 to 941 U mg(-1) for syringaldazine, from 18 to 1565 U mg(-1) for ABTS, and from 4 to 44 U mg(-1) for guaiacol. The apparent affinity constants (KM) also showed significant differences between the different strains, from 12 to 52 micromol l(-1) for syringaldazine, from 8 to 79 micromol l(-1) for ABTS, and from 0.46 to 6.61 mmol l(-1) for guaiacol. No differences were found either on the effect of increasing concentrations of organic solvent (acetonitrile) or on the activity pH profile. The temperature profile was the same for all the P. ostreatus strains, except for the IE8 strain, which seems to be more sensitive to temperature. The kinetic and stability data from the six P. ostreatus strains were also compared with those obtained from other white rot fungi, Coriolopsis gallica and Trametes versicolor, showing clear differences. CONCLUSION: The different P. ostreatus isolates showed different kinetic constants. SIGNIFICANCE AND IMPACT OF THE STUDY: The different enzymatic properties of laccases from various P. ostreatus strains should be considered for a potential industrial or environmental application.

Effect of growth conditions on the production of manganese peroxidase by three strains of Bjerkandera adusta.

We were looking for a strain of Bjerkandera adusta that produces high titres of manganese peroxidase under optimal conditions for large-scale enzyme purification. We have chosen two strains from the University of Alberta Microfungus Collection and Herbarium, UAMH 7308 and 8258, and compared the effects of growth conditions and medium composition on enzyme production with the well-characterized strain BOS55 (ATCC 90940). Of four types of cereal bran examined, rice bran at 3% (w/v) in 60 mM phosphate buffer pH 6 supported the highest levels of enzyme production. Using 100 mL medium in 500-mL Erlenmeyer flasks, maximum enzyme levels in the culture supernatant occurred after about 10 days of growth; 5.5 U x mL(-1) for UAMH 7308, 4.4 U x mL(-1) for UAMH 8258, and 1.7 U x mL(-1) for BOS55, where units are expressed as micromoles of Mn-malonate formed per minute. Growth as submerged cultures in 10-L stirred tank reactors produced 3.5 U x mL(-1) of manganese peroxidase (MnP) by UAMH 8258 and 2.5 U x mL(-1) of MnP by 7308, while enzyme production by BOS55 was not successful in stirred tank reactors but could be scaled up in 2-L shake flasks containing 400 mL rice bran or glucose-malt-yeast extract (GMY)-Mn-glycolate medium to produce MnP levels of 1.7 U x mL(-1). These results show that the two strains of B. adusta, UAMH 7308 and 8258, can produce between two and three times the manganese peroxidase level of B. adusta BOS55, that they are good candidates for scale up of enzyme production, and that the rice bran medium supports higher levels of enzyme production than most previously described media.

Enzyme conjugation to the polysaccharide chitosan: smart biocatalysts and biocatalytic hydrogels.

Laccase from Coriolopsis gallica was conjugated to the renewable biopolymer chitosan using carbodiimide chemistry. The laccase-chitosan conjugate was observed to offer three unique properties. First, the laccase-chitosan conjugate displayed pH-responsive behavior such that the conjugate was soluble and active under acidic conditions, but precipitated when the pH was raised toward neutrality. Second, the laccase-chitosan conjugate was more stable than free laccase at extreme pHs. At pH 1, the inactivation rate constant (k(in)) for the soluble laccase-chitosan conjugate was 20-fold less than that for free laccase. At pH 13, k(in) for the insoluble laccase-chitosan conjugate was nearly 3-fold less than that for free laccase. Finally, the laccase-chitosan conjugate could be cross-linked under mild conditions to create biocatalytic hydrogels. Potential benefits for enzyme-chitosan conjugates are discussed.

Chemical modification of hemoglobin improves biocatalytic oxidation of PAHs.

Chemical modifications on human hemoglobin were performed with the aim to change both surface and active-site hydrophobicities. The modifications included covalent coupling of poly(ethylene)glycol (5000 MW) on free amino groups and the methyl esterification of free carboxylic groups. The modified hemoglobin was assayed for the oxidation of 11 polycyclic aromatic hydrocarbons (PAHs) and 2 organosulfur aromatic compounds. Acenaphthene, anthracene, azulene, benzo(a)pyrene, fluoranthene, fluorene, phenanthrene, and pyrene were transformed to their respective quinones, while for chrysene and biphenyl no biocatalytic reaction could be detected. Dibenzothiophene and thianthrene were oxidized to form sulfoxides. The doubly modified hemoglobin, PEG-Met-hemoglobin, showed up to 10 times higher activity than the unmodified protein. The kinetic constants show that the PEG-Met-hemoglobin has a significantly higher catalytic efficiency. The equilibrium substrate binding constants for unmodified and PEG-Met-modified hemoglobis and hemoglobin show that this catalytic enhancement could be attributed to the affinity increase for hydrophobic substrates in the modified protein.

A cytochrome c variant resistant to heme degradation by hydrogen peroxide.

BACKGROUND: Cytochrome c has peroxidase-like activity and can catalyze the oxidation of a variety of organic substrates, including aromatic, organosulfur and lipid compounds. Like peroxidases, cytochrome c is inactivated by hydrogen peroxide. During this inactivation the heme prosthetic group is destroyed. RESULTS: Variants of the iso-1-cytochrome c were constructed by site-directed mutagenesis and were found to be more stable in the presence of hydrogen peroxide than the wild type. No heme destruction was detected in a triple variant (Tyr67-->Phe/Asn52-->Ile/Cys102-->Thr) with the catalytic hydrogen peroxide concentration of 1 mM, even following the loss of catalytic activity, whereas both double variants Tyr67-->Phe/Cys102-->Thr and Asn52-->Ile/Cys102-->Thr showed a greater rate of peroxide-induced heme destruction than observed with the wild-type protein. CONCLUSIONS: Heme destruction and catalytic inactivation are two independent processes. An internal water molecule (Wat166) is shown to be important in the heme destruction process. The absence of a protein radical in the resistant variant suggests that the protein radical is necessary in the heme destruction process, but presumably is not involved in the reactions leading up to the protein inactivation.

Polycyclic aromatic hydrocarbon metabolism by white rot fungi and oxidation by Coriolopsis gallica UAMH 8260 laccase.

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 microg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from Coriolopsis gallica. The reaction mixtures contained 20 microM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene. There was no clear relationship between the ionization potential of the substrate and the first-order rate constant (k) for substrate loss in vitro in the presence of ABTS. The effects of mediating substrates were examined further by using anthracene as the substrate. Hydroxybenzotriazole (HBT) (1 mM) supported approximately one-half the anthracene oxidation rate (k = 2.4 h(-1)) that ABTS (1 mM) supported (k = 5.2 h(-1)), but 1 mM HBT plus 1 mM ABTS increased the oxidation rate ninefold compared with the oxidation rate in the presence of ABTS, to 45 h(-1). Laccase purified from Pleurotus ostreatus had an activity similar to that of C. gallica laccase with HBT alone, with ABTS alone, and with 1 mM HBT plus 1 mM ABTS. Mass spectra of products obtained from oxidation of anthracene and acenaphthene revealed that the dione derivatives of these compounds were present.

The branched-chain dodecylbenzene sulfonate degradation pathway of Pseudomonas aeruginosa W51D involves a novel route for degradation of the surfactant lateral alkyl chain.

Pseudomonas aeruginosa W51D is able to grow by using branched-chain dodecylbenzene sulfonates (B-DBS) or the terpenic alcohol citronellol as a sole source of carbon. A mutant derived from this strain (W51M1) is unable to degrade citronellol but still grows on B-DBS, showing that the citronellol degradation route is not the main pathway involved in the degradation of the surfactant alkyl moiety. The structures of the main B-DBS isomers and of some intermediates were identified by gas chromatography-mass spectrometric analysis, and a possible catabolic route is proposed.

Industrial dye decolorization by laccases from ligninolytic fungi.

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized.