Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System.

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Selection of biosurfactan/bioemulsifier-producing bacteria from hydrocarbon-contaminated soil.

Petroleum-derived hydrocarbons are among the most persistent soil contaminants, and some hydrocarbon-degrading microorganisms can produce biosurfactants to increase bioavailability and degradation. The aim of this work was to identify biosurfactant-producing bacterial strains isolated from hydrocarbon-contaminated sites, and to evaluate their biosurfactant properties. The drop-collapse method and minimal agar added with a layer of combustoleo were used for screening, and positive strains were grown in liquid medium, and surface tension and emulsification index were determined in cell-free supernantant and cell suspension. A total of 324 bacterial strains were tested, and 17 were positive for the drop-collapse and hydrocarbon-layer agar methods. Most of the strains were Pseudomonas, except for three strains (Acinetobacter, Bacillus, Rhodococcus). Surface tension was similar in cell-free and cell suspension measurements, with values in the range of 58 to 26 (mN/m), and all formed stable emulsions with motor oil (76-93% E24). Considering the variety of molecular structures among microbial biosurfactants, they have different chemical properties that can be exploited commercially, for applications as diverse as bioremediation or degradable detergents.

Microbiological Impact of the Use of Reclaimed Wastewater in Recreational Parks.

Reclaimed wastewater for irrigation is an opportunity for recovery of this natural resource. In this study, microbial risk from the use of treated wastewater for irrigation of recreational parks in the city of Chihuahua, evaluating the effect of distribution distance, season, and presence of storage tanks, was analyzed. Escherichia coli, Salmonella, and multidrug-resistant bacteria were recovered from samples of reclaimed water and soils at recreational parks in Chihuahua by the membrane filtration method, using selected agars for microbial growth. Samples were taken at three different seasons. No correlation in the presence of microbial indicators and multidrug-resistant bacteria (p > 0.05) was found between the distance from the wastewater treatment plant to the point of use. Presence of storage tanks in parks showed a significant effect (p < 0.05) with a higher level of E. coli. The highest count in wastewater occurred in summer. We isolated 392 multidrug-resistant bacteria from water and soil; cluster analysis showed that the microorganisms at each location were of different origins. Irrigation with reclaimed wastewater did not have a negative effect on the presence of microbial indicators of the quality of soils in the parks. However, the prevalence of multidrug-resistant bacteria still represents a potential risk factor for human health.

Oxidative damage to Pseudomonas aeruginosa ATCC 27833 and Staphylococcus aureus ATCC 24213 induced by CuO-NPs.

The cytotoxicity of nanoparticles (NPs) and their properties are important issues in nanotechnology research. Particularly, NPs affect the metabolism of microorganisms due to NP interactions with some biomolecules. In order to assess the mechanisms underlying NPs toxicity, we studied the damage caused by copper oxide nanoparticles (CuO-NPs) on Staphylococcus aureus ATCC 24213 and Pseudomonas aeruginosa ATCC 27833. Spherical CuO-NPs characterized by their diameter (13 ± 3 nm) were synthesized with a maximum of 254 nm. These NPs reduced cell viability, with a minimum inhibitory concentration (MIC) of 500 and 700 ppm for Staphylococcus aureus and Pseudomonas aeruginosa, respectively. Surfactant was added to reduce the NP agglomeration, but it did not present any effect. The mechanism of CuO-NPs as antimicrobial agent was assessed by analyzing solubilized Cu2+, quantifying DNA release in the culture media, and measuring intracellular reactive oxygen species (ROS). CuO-NPs induced severe damage on cells as revealed by confocal optical microscopy and scanning electron microscopy (SEM). Our results indicated that CuO-NPs interacted with bacteria, triggering an intracellular signaling network which produced oxidative stress, leading to ROS generation. Finally, we concluded that CuO-NPs exhibited higher antibacterial activity on Gram-negative bacteria than on Gram-positive ones.

Selection of biosurfactan/bioemulsifier-producing bacteria from hydrocarbon-contaminated soil

Petroleum-derived hydrocarbons are among the most persistent soil contaminants, and some hydrocarbon-degrading microorganisms can produce biosurfactants to increase bioavailability and degradation. The aim of this work was to identify biosurfactant-producing bacterial strains isolated from hydrocarbon-contaminated sites, and to evaluate their biosurfactant properties. The drop-collapse method and minimal agar added with a layer of combustoleo were used for screening, and positive strains were grown in liquid medium, and surface tension and emulsification index were determined in cell-free supernantant and cell suspension. A total of 324 bacterial strains were tested, and 17 were positive for the drop-collapse and hydrocarbon-layer agar methods. Most of the strains were Pseudomonas, except for three strains (Acinetobacter, Bacillus, Rhodococcus). Surface tension was similar in cell-free and cell suspension measurements, with values in the range of 58 to 26 (mN/m), and all formed stable emulsions with motor oil (76-93 percent E24). Considering the variety of molecular structures among microbial biosurfactants, they have different chemical properties that can be exploited commercially, for applications as diverse as bioremediation or degradable detergents.