Polyamide 6/chitosan nanofibers as support for the immobilization of Trametes versicolor laccase for the elimination of endocrine disrupting chemicals

In recent years, there has been an increase in efforts to improve wastewater treatment as the concentration of dangerous pollutants, such as endocrine disrupting chemicals, in wastewater increases. These compounds, which mimic the effect of hormones, have a negative impact on human health and are not easily removed from water. One way to effectively eliminate these pollutants is to use enzymatically activated materials. In this study, we report on the use of laccase from the white rot fungus Trametes versicolor immobilized onto polyamide 6/chitosan (PA6/CHIT) nanofibers modified using two different spacers (bovine serum albumin and hexamethylenediamine). We then tested the ability of the PA6/CHIT-laccase biocatalysts to eliminate a mixture containing 50 μM of two endocrine disrupting chemicals: bisphenol A and 17α-ethinylestradiol. The PA6/CHIT nanofiber matrix used in this study not only proved to be a suitable carrier for immobilized and modified laccase but was also efficient in the removal of a mixture of endocrine disrupting chemicals in three treatment cycles.     

Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.     

Utilization of cross‐linked laccase aggregates in a perfusion basket reactor for the continuous elimination of endocrine‐disrupting chemicals

A perfusion basket reactor (BR) was developed for the continuous utilization of insolubilized laccase as cross‐linked enzyme aggregates (CLEAs). The BR consisted of an unbaffled basket made of a metallic filtration module filled with CLEAs and continuously agitated by a 3‐blade marine propeller. The agitation conditions influenced both the apparent laccase activity in the reactor and the stability of the biocatalyst. Optimal laccase activity was obtained at a rotational speed of 12.5 rps and the highest stability was reached at speeds of 1.7 rps or lower. The activity and stability of the biocatalyst were affected drastically upon the appearance of vortices in the reaction medium. This reactor was used for the continuous elimination of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA), and triclosan (TCS). Optimization of EDC elimination by laccase CLEAs as a function of temperature and pH was achieved by response surface methodology using a central composite factorial design. The optimal conditions of pH and temperature were, respectively, 4.8 and 40.3°C for the elimination of p353NP (a branched isomer of NP), 4.7 and 48.0°C for BPA, and 4.9 and 41.2°C for TCS. Finally, the BR was used for the continuous elimination of these EDCs from a 5 mg L^?1 aqueous solution using 1 mg of CLEAs at pH 5 and room temperature. Our results showed that at least 85% of these EDCs could be eliminated with a hydraulic retention time of 325 min. The performances of the BR were quite stable over a 7‐day period of continuous treatment. Furthermore, this system could eliminate the same EDCs from a 100 mg L^?1 solution. Finally, a mathematical model combining the Michaelis–Menten kinetics of the laccase CLEAs and the continuous stirred tank reactor behavior of the BR was developed to predict the elimination of these xenobiotics. Biotechnol. Bioeng. 2009;102: 1582–1592. © 2008 Wiley Periodicals, Inc.     

Conjugation of laccase from the white rot fungus Trametes versicolor to chitosan and its utilization for the elimination of triclosan

A commercial laccase from Trametes versicolor was conjugated with biopolymer chitosan using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as the cross-linking agent. Laccase-chitosan conjugation strategies were tested using different molar ratios of glucosamine monomer/protein with different molar excess ratios of EDC relative to laccase. Immobilization techniques were developed to improve the stability against thermal and chemical denaturation, storage and reusability of this biocatalyst. The conjugation resulted in a solid biocatalyst with an apparent laccase activity of ±626 U/g, 12 and 60 folds higher in the conjugation efficiency of biocatalyst relative to the immobilized and free laccase activity respectively when compared with zero EDC/laccase ratio used in conjugation solution. The conjugated laccases formed successfully eliminated the emerging pollutant triclosan (TCS) from aqueous solutions, having a higher potential to transform TCS than free laccase. UPLC-QTOF results indicate the formation of TCS oligomers. Furthermore, they are the first evidence of direct dechlorination of TCS mediated by the oxidative action of laccases.     

Removal and recovery of uranium from aqueous solutions by Ca-alginate immobilized Trichoderma harzianum

The ability of Ca-alginate immobilized Trichoderma harzianum has been explored for removal and recovery of uranium from aqueous streams. Ca-alginate as polymeric support was selected after screening different matrices. Immobilization of Trichoderma harzianum to Ca-alginate improved the stability as well as uranium biosorption capacity of biosorbent at 28 ± 2 °C and 200 rpm. The suitability of packed bed column operations was illustrated by obtaining break through curves at different bed heights, flow rates and inlet uranium concentrations. The adsorption column containing 1.5 g dry weight of immobilized material has purified 8.5 L of bacterial leach liquor (58 mg/L U) before break through occurred and the biosorbent became saturated after 25 L of influent. Sorbed uranium was recovered in 200 ml of 0.1 N HCl resulting in 98.1–99.3% elution by 0.1 N HCl, which regenerated the biosorbent facilitating the sorption–desorption cycles for better economic feasibility without any significant alteration in sorption capacity/elution efficiency.     

Formation of coniferyl alcohol from ferulic acid by the white rot fungus Trametes

The white rot fungus, Trametes sp., was cultivated in a medium containing ferulic acid, glucose and ethanol under aerobic conditions in submerged culture. The ferulic acid was transformed into coniferyl alcohol, coniferylaldehyde, dihydroconiferyl alcohol, vanillic acid, vanillyl alcohol, 2-methoxyhydroquinone and 2-methoxyquinone during 48–120 hr of cultivation. The amount of coniferyl alcohol in the culture reached a maximum after 90 hr with ca 40% of the initial amount of ferulic acid. Cinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, p -coumaric acid and sinapic acid were also transformed into the corresponding alcohols, benzoic acids and benzyl alcohols in the fungus culture.     

Purification of pyranose oxidase from the white rot fungus Irpex lacteus and its cooperation with laccase in lignin degradation

Laccase and peroxidases mainly cause polymerization of lignin in vitro due to the random coupling of the phenoxy radicals or quinoid intermediates. White rot fungi may avoid polymerization in vivo by reduction of these intermediates. Pyranose oxidase is suggested to play such a role based on its quinone-reducing activity, but direct evidence has been lacking. In this study, a pyranose oxidase was purified from the white rot fungus Irpex lacteus and partially characterized. The enzyme is composed of four subunits of 71 kDa as determined by SDS-PAGE. It exhibits maximum activity at pH 6.5 and 55 °C and is rather stable. d-glucose is the preferred substrate, but d-galactose, l-sorbose and d-xylose are also readily oxidized. In addition to O₂, the enzyme can also transfer electrons to various quinones and the ABTS [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] cation radical. Laccase-generated quinoids are also reduced by the enzyme. Four different technical lignins were treated with laccase with and without pyranose oxidase. Subsequent gel permeation chromatography analysis demonstrated that the pyranose oxidase efficiently inhibited the polymerization of lignin caused by laccase and even brought about degradation.     

Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants

A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu(2+) centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50 degrees C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, -20 degrees C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit((R))C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25 degrees C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.     

Immobilization of laccase by encapsulation in a sol-gel matrix and its characterization and use for the removal of estrogens

Laccase from Myceliophthora thermophila was immobilized by encapsulation in a sol-gel matrix based on methyltrimethoxysilane and tetramethoxysilane. The amount of laccase used for the preparation of the hydrogel was in the range 2.2-22 mg of protein/mL sol and the corresponding enzymatic activities were in the range 5.5-17.0 U/g biocatalyst. The kinetic parameters of the encapsulated laccase showed that the immobilized enzyme presented lower affinity for the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). However, the stability of laccase was significantly enhanced after immobilization; thus, both pH and thermal stability improved about 10-30% and tolerance to different inactivating agents (NaN(3) , ZnCl(2) , CoCl(2) , CaCl(2) , methanol, and acetone) was 20-40% higher. The reusability of the immobilized laccase was demonstrated in the oxidation of ABTS for several consecutive cycles, preserving 80% of the initial laccase activity after 10 cycles. The feasibility of the immobilized biocatalyst was tested for the continuous elimination of Acid Green 27 dye as a model compound in a packed-bed reactor (PBR). Removals of 70, 58, 57, and 55% were achieved after four consecutive cycles with limited adsorption on the support: only 10-15%. Finally, both batch stirred tank reactor (BSTR) operated in several cycles and PBR, containing the solid biocatalyst were applied for the treatment of a solution containing the endocrine disrupting chemicals (EDCs): estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2). Eliminations of EDCs in the BSTR were higher than 85% and the reusability of the biocatalyst for the degradation of those estrogens was demonstrated. In the continuous operation of the PBR, E1 was degraded by 55% and E2 and EE2 were removed up to 75 and 60%, at steady-state conditions. In addition, a 63% decrease in estrogenic activity was detected.     

Cloning and expression of ecdysone receptor (EcR) from the intertidal copepod, Tigriopus japonicus

Ecdysteroids are steroid hormones that play an important role in development, growth, molting of larva, and reproduction in the Arthropoda. The effect of ecdysteroids is mediated by its binding to ecdysteroid receptor (EcR). To investigate the role of EcR during development and the effect to environmental stressors on EcR expression in a copepod, we isolated and characterized cDNA and 5′-promoter region of the Tigriopus japonicus EcR (TJ-EcR), and studied mRNA expression pattern. The full-length TJ-EcR cDNA sequence was 1962 bp in length and the open reading frame encoded 546 amino acids. The deduced TJ-EcR protein contained well-conserved DNA-binding domain and ligand-binding domain. Phylogenetic analysis revealed that TJ-EcR was clustered with the EcR of other crustaceans. TJ-EcR mRNA was expressed in a developmental stage-specific manner: high in early developmental stages and low in the adult stage. Significantly elevated expression of the TJ-EcR gene in adults was detected at hypersalinity (42 ppt) and high temperature (35 °C) condition. The 5′-flanking region of TJ-EcR gene contains heat shock protein 70 response elements, implying that the environmental stressors may affect its expression via the stress-sensor. In addition, bisphenol A (100 µg/L) repressed TJ-EcR expression. Our results suggest that TJ-EcR could be a biomarker for the monitoring of the impact of environmental stressors in copepods.     

Comparative endocrine disruptive effects of contaminants in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea

We investigated variables related to thyroid, vitamin A and calcitriol homeostasis, immune function and tumour development in ringed seals (Phoca hispida) from the polluted Baltic Sea and a less polluted reference location at Svalbard, Norway. We also examined the relationships between the biological variables and the concentrations of persistent organic pollutants (POPs) and their hydroxylated (OH) metabolites. Our data show higher plasma concentrations of free triiodothyronine (T3), and ratios of free and total T3 in Baltic seals as compared to Svalbard seals. Baltic seals had also higher hepatic mRNA expressions of deiodinase-I, thyroid hormone receptor β, retinoic acid receptor α, growth hormone receptor and interleukin-1β compared to Svalbard seals. Levels of plasma retinol were lower in the Baltic seals as compared to Svalbard seals. No geographical difference was observed for other thyroid hormone levels and hepatic retinoid levels. Ratios of free and total T3 were positively correlated to OH-POPs in plasma. The results of the present study suggest that endocrine homeostasis may be affected by contaminant and metabolite exposure in the Baltic ringed seals with respect to circulating hormones and retinol and hepatic mRNA expressions. In addition, OH-POPs may putatively produce the disruption of thyroid hormone transport in plasma.     

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co²⁺ rather than Zn²⁺: the k_cat (s⁻¹) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co²⁺, and Zn²⁺ conditions, respectively. Consistently, addition of low concentrations of Co²⁺ to PaAP previously saturated with Zn²⁺ greatly enhanced the enzymatic activity, suggesting that Co²⁺ may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co²⁺ or Zn²⁺ commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co²⁺- and Zn²⁺-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co²⁺ for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.     

Improved protocol for isolation of Campylobacter spp. from retail broiler meat and use of pulsed field gel electrophoresis for the typing of isolates

To improve the detection of Campylobacter spp. in retail broiler meat, a reference method (R subsamples) based on the enrichment of 25 g of meat in Bolton broth at 42 °C under microaerobiosis was compared with an alternative method (A subsamples) consisting in the rinsing of meat samples for 30 s in buffered peptone water with antimicrobials with incubation at 42 °C under aerobiosis. One piece of meat (breasts, tenderloins and thighs) was rinse in experiment 1 (A1) and two pieces in experiment 2 (A2). Campylobacter spp. were isolated on agar plates and identified by PCR. Retail samples in Alabama had less prevalence (P ≤ 0.05) than samples in the state of Washington. The percentage of positive was higher (P ≤ 0.05) in A than in R subsamples and rinsing two pieces of meat yielded the highest percentage of positive subsamples. R subsamples showed variations in the prevalence by product. However, A subsamples had similar prevalence of positives among products compare to the result from reference method. More Campylobacter coli isolates were collected in A2 subsamples. Pulse field gel electrophoresis (PFGE) was used as subtyping method to study the genome similarity among the isolates from all methods. A larger diversity of isolates were detected by PFGE in A2 subsamples. Denaturing gradient gel electrophoresis analysis suggested that the initial bacterial populations of the meat samples impact the final bacterial profile after enrichment. Rinsing broiler meats was less time consuming, required less sample preparation and was more sensitive than the reference method for the isolation of naturally occurring Campylobacter spp. This new method could help with epidemiological and intervention studies to control Campylobacter spp.     

Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR

Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4 × 10⁴ target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10³ copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R² ≥ 0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters.     

Harnessing the power of enzymes for environmental stewardship

Enzymes are versatile catalysts with a growing number of applications in biotechnology. Their properties render them also attractive for waste/pollutant treatment processes and their use might be advantageous over conventional treatments. This review highlights enzymes that are suitable for waste treatment, with a focus on cell-free applications or processes with extracellular and immobilized enzymes. Biological wastes are treated with hydrolases, primarily to degrade biological polymers in a pre-treatment step. Oxidoreductases and lyases are used to biotransform specific pollutants of various nature. Examples from pulp and paper, textile, food and beverage as well as water and chemical industries illustrate the state of the art of enzymatic pollution treatment. Research directions in enzyme technology and their importance for future development in environmental biotechnology are elaborated. Beside biological and biochemical approaches, i.e. enzyme prospection and the design of enzymes, the review also covers efforts in adjacent research fields such as insolubilization of enzymes, reactor design and the use of additives. The effectiveness of enzymatic processes, especially when combined with established technologies, is evident. However, only a limited number of enzymatic field applications exist. Factors like cost and stability of biocatalysts need to be addressed and the collaboration and exchange between academia and industry should be further strengthened to achieve the goal of sustainability.