Staphylococcus aureus microbial biofilms degradation using cellobiose dehydrogenase from Thermothelomyces thermophilus M77.

This work reports biochemical characterization of Thermothelomyces thermophilus cellobiose dehydrogenase (TthCDHIIa) and its application as an antimicrobial and antibiofilm agent. We demonstrate that TthCDHIIa is thermostable in different ionic solutions and is capable of oxidizing multiple mono and oligosaccharide substrates and to continuously produce H2O2. Kinetics measurements depict the enzyme catalytic characteristics consistent with an Ascomycota class II CDH. Our structural analyses show that TthCDHIIa substrate binding pocket is spacious enough to accommodate larger cello and xylooligosaccharides. We also reveal that TthCDHIIa supplemented with cellobiose reduces the viability of S. aureus ATCC 25923 up to 32 % in a planktonic growth model and also inhibits its biofilm growth on 62.5 %. Furthermore, TthCDHIIa eradicates preformed S. aureus biofilms via H2O2 oxidative degradation of the biofilm matrix, making these bacteria considerably more susceptible to gentamicin and tetracycline.

A non-waste strategy for enzymatic hydrolysis of cellulose recovered from domestic wastewater.

Cellulose is a potential resource to be recovered from wastewater treatment plants (WWTP). Enzyme formulations can be employed to hydrolyze cellulose into fermentable sugars, to be further used as biochemical building blocks or reducing its recalcitrance to further treatment processes. This study proposed the production, recovery and formulation of cellulase using domestic wastewater as culture medium and its application for the hydrolysis of cellulosic residues recovered from WWTPs. Cellulose was recovered from raw sanitary wastewater using a fine-mesh sieve (0.35 mm) and quantified through enzymatic hydrolysis and thermogravimetric analysis. The production, concentration and formulation of cellulase enzyme resulted in an enzymatic blend of endoglucanases (7.3 UFP/mL), cellobiohydrolases (7.4 UCMC/mL) and beta-glucosidases (4.4 UBGL/mL). The content of the recovered cellulosic material was 21.3% according to enzymatic hydrolysis and 27.7 for thermogravimetric results. The enzymatic hydrolysis of the WWTP residue using the produced cellulase (107.6 ± 10.2 mgreduc/gresidue) showed better results than using the commercial cellulase complex (66.4 ± 2.5 mgreduc/gresidue). This fact showed the potential of application of the produced enzyme for the hydrolysis of cellulosic residues recovered from WWTP processes. In a non-waste biorefinery approach, the generated hydrolysate can be further used for producing added-value biomolecules including biofuels and biochemicals.

Ionic Cross-Linking as a Strategy to Modulate the Properties of Oral Mucoadhesive Microparticles Based on Polysaccharide Blends.

Polymer blends of gellan gum (GG)/retrograded starch(RS) and GG/pectin (P) were cross-linked with calcium, aluminum, or both to prepare mucoadhesive microparticles as oral carriers of drugs or nano systems. Cross-linking with different cations promoted different effects on each blend, which can potentially be explored as novel strategies for modulating physical-chemical and mucoadhesive properties of microparticles. Particles exhibited spherical shapes, diameters from 888 to 1764 µm, and span index values lower than 0.5. Blends of GG:P cross-linked with aluminum resulted in smaller particles than those obtained by calcium cross-linking. GG:RS particles exhibited larger sizes, but cross-linking this blend with calcium promoted diameter reduction. The uptake rates of acid medium were lower than phosphate buffer (pH 6.8), especially GG:RS based particles cross-linked with calcium. On the other hand, particles based on GG:P cross-linked with calcium absorbed the highest volume of acid medium. The percentage of systems erosion was higher in acid medium, but apparently occurred in the outermost layer of the particle. In pH 6.8, erosion was lower, but caused expressive swelling of the matrixes. Calcium cross-linking of GG:RS promoted a significantly reduction on enzymatic degradation at both pH 1.2 and 6.8, which is a promising feature that can provide drug protection against premature degradation in the stomach. In contrast, GG:P microparticles cross-linked with calcium suffered high degradation at both pH values, an advantageous feature for quickly releasing drugs at different sites of the gastrointestinal tract. The high mucoadhesive ability of the microparticles was evidenced at both pH values, and the Freundlich parameters indicated stronger particle-mucin interactions at pH 6.8.

Computational studies of polyurethanases from Pseudomonas.

Polyurethanes (PU) are multifunctional polymers, used in automotive industry, in coatings, rigid and flexible foams, and also in biomimetic materials. In the same way as all plastic waste, the incorrect disposal of these materials leads to the accumulation of polyurethanes in the environment. To reduce the amount of waste as well as add value to degradation products, bioremediation methods have been studied for waste management of PU. Enzymes of the hydrolases class have been experimentally tested for enzymatic degradation of PU, with very promising results. In this work, two enzymes that can degrade polyurethanes were studied by molecular dynamics simulations: a protease and an esterase, both from Pseudomonas. From molecular dynamics simulations analysis, it was observed the stability of the structures, both in the simulations of the free enzymes and in the simulations of the complexes with a PU monomer. Hydrogen bonds were formed with the monomer and the enzymes throughout the simulation time, and the interaction free energy was found to be strongly negative, pointing to strong interactions in both cases.

Toxicity of enzymatically decolored textile dyes solution by horseradish peroxidase.

The oxidative systems including enzymatic systems have been widely studied as an alternative for textile effluents treatment. However, studies have shown that some oxidative processes can produce degradation products with higher toxicity than the untreated dye. In this work, enzymatic dye decolorization was evaluated by horseradish peroxidase enzyme (HRP) and the toxicity of discoloration products was evaluate against Daphnia magna, Euglena gracilis algae, and Vibrio fischeri. Dye decolorization kinetics data were evaluated and the pseudo-second-order model showed the best-fitting to the experimental data. In addition, it was observed an increased acute and chronic toxicity associated with the decolorization efficiency. The Reactive Blue 19 and Reactive Black dye showed the highest toxicity against D. Magna (16 toxicity factor) and V. Fischeri (32 toxicity factor) after enzymatic decolorization. For the chronic toxicity against D. Magna, Reactive Red was the only dye with no fertility inhibition. In relation to toxicity tests with E. gracilis algae, it was not observed photosynthetic inhibition for all dyes. This study verified the viability of the enzyme horseradish peroxidase in the textile dyes decolorization and the importance to evaluate the decolorization products.

Potentialities of active membranes with immobilized laccase for Bisphenol A degradation.

Herein, we report the development of immobilized laccase based membrane bioreactor as a novel bio-catalytic system for the degradation of emerging endocrine disruptor i.e., Bisphenol A. Two laccase forms i.e. (1) in-house isolated and purified from an indigenous white-rot fungi Pycnoporus sanguineus (CS43) and (2) Trametes versicolor (commercial laccase from Sigma-Aldrich®) were immobilized on a multi-channel ceramic membrane (1.4µm in diameter) using 4% glutaraldehyde as a cross-linking agent. The immobilization yield and bisphenol A degradation activities of immobilized laccases were recorded at various pH levels. The surface topographies of immobilized-laccase membranes were accessed by scanning electron microscopy. In this study, 100% degradation of bisphenol A (20mg/L) was achieved in less than 24h in the presence of laccase from P. sanguineus (CS43) (620.55±14.85U/L) and T. versicolor (620.55±14.85U/L). The enzymes showed an optimal activity at pH 5 and 5.4 with a degradation rate of 204.8±1.8 and 79.0±0.1µmol/min/U for P. sanguineus (CS43) and T. versicolor, respectively. In conclusion, the highest immobilization of unit per square centimeter and efficient degradation potentiality strongly recommend the newly developed immobilized laccase based membrane bioreactor as a novel tool to tackle emerging contaminants degradation issues.

Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment

Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1-6 percent NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 percent protein removal at 6 h duration and 2 percent (v/v) protease addition was found to be the optimum dosage value.

Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment.

Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1-6 % NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and 2 % (v/v) protease addition was found to be the optimum dosage value.

Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment

Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1-6 % NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and 2 % (v/v) protease addition was found to be the optimum dosage value.