ABSTRACT A three-level Box-Behken factorial design combined with the response surface methodology... more ABSTRACT A three-level Box-Behken factorial design combined with the response surface methodology (RSM) was applied as a tool to study the laccase-catalyzed removal of three estrogenic compounds: estrone (E1), estradiol (E2) and ethinylestradiol (EE2), in a continuous enzymatic membrane reactor (EMR). Three main factors affecting the treatment efficiency were considered: enzyme activity, hydraulic residence time (HRT) and oxygenation rate. As expected, laccase activity and HRT showed great effects and, interestingly, it was demonstrated the relevance of the oxygen in the improvement of oxidation reaction kinetics through the rise of the oxygen dissolved above saturation levels. When considering elimination rates as response, optimal conditions were: 1,000 U/L of laccase, 1 h HRT and 60 mgO2/(L•h) of oxygenation rate, predicting 2.82-3.24 mg eliminated/(L•h), which meant 71-81% of oxidation. These optimum conditions were successfully validated, and a 75% of estrogenicity reduction was achieved. On the other hand, only 100 U/L were found as optimal to maximize the efficacy of the enzyme: E1 was oxidized by 0.06 mg/(L•h•U), although the removal of estrogenicity decreased to 60%. The methodology was also applied to maximize the reduction of estrogenic activity: the highest values assayed (1,000 U/L, HRT 4 h and 60 mgO2/(L•h)) provided a 99% of detoxification.
Continuous production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an... more Continuous production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an Expanded-Bed Bioreactor was successfully achieved for more than 30 days of operation. The stability in continuous production was attained because of two modifications in the operational policy: an adequate feed control and the supply of oxygen in a pulsing mode. Feed control allowed to stablish an equilibrium between primary and secondary metabolism, thus avoiding metabolic stress. The use of pulsation had two beneficial effects: 1) Increase of stability by avoiding bed compactation during operation performance; 2) Control of mycelial pellet size. The bioreactor operated without oxygen pulsation did not maintain enzyme production longer than 14 days.
Environmental Science and Pollution Research, Oct 21, 2015
Enzymes immobilization is a useful way to allow enzyme reuse and increase their stability. A high... more Enzymes immobilization is a useful way to allow enzyme reuse and increase their stability. A high redox potential laccase from Trametes versicolor (TvL) and a low redox potential, but commercially available low-cost laccase from Myceliophthora thermophila (MtL), were successfully immobilized and co-immobilized onto fumed silica nanoparticles (fsNP). Enzyme loads of 1.78 ± 0.07, 0.69 ± 0.03, and 1.10 ± 0.01 U/mg fsNP were attained for the optimal doses of TvL, MtL, and co-immobilized laccases, respectively. In general, the laccase-fsNP conjugates showed a higher resistance against an acidic pH value (i.e., pH 3), and a higher storage stability than free enzymes. In addition, immobilized enzymes exhibited a superior long-term stability than free laccases when incubated in a secondary effluent from a municipal wastewater treatment plant (WWTP). For instance, the residual activity after 2 weeks for the co-immobilized laccases and the mixture of free laccases were 40.2 ± 2.5 % and 16.8 ± 1.0 %, respectively. The ability of the laccase-fsNP to remove a mixture of (14)C-bisphenol A (BPA) and (14)C-sodium diclofenac (DCF) from spiked secondary effluents was assessed in batch experiments. The catalytic efficiency was highly dependent on both the microbial source and state of the biocatalyst. The high redox potential TvL in free form attained a four-fold higher percentage of BPA transformation than the free MtL. Compared to free laccases, immobilized enzymes led to much slower rates of BPA transformation. For instance, after 24 h, the percentages of BPA transformation by 1000 U/L of a mixture of free laccases or co-immobilized enzymes were 67.8 ± 5.2 and 27.0 ± 3.9 %, respectively. Nevertheless, the use of 8000 U/L of co-immobilized laccase led to a nearly complete removal of BPA, despite the unfavorable conditions for laccase catalysis (pH ~ 8.4). DCF transformation was not observed for any of the enzymatic systems, showing that this compound is highly recalcitrant toward laccase oxidation under realistic conditions.
The enzyme-mediated polymerization of bioactive phenolic compounds, such as the flavonoid rutin, ... more The enzyme-mediated polymerization of bioactive phenolic compounds, such as the flavonoid rutin, has gained interest due to the enhanced physico-chemical and biological properties of the products, which increases their potential application as a nutraceutical. In this work, the influence of enzyme activity on rutin oligomerization was evaluated in reactions with low (1000 U/L) and high (10,000 U/L) initial laccase activities. For both reactions, high molecular weight oligomer fractions showed better properties compared to lower weight oligomers. Products of the reaction with low laccase activity exhibited thermal stability and antioxidant potential similar to control reaction, but led to higher inhibitory activity of xanthine oxidase and apparent aqueous solubility. Oligomers obtained in the reaction with high laccase activity showed better apparent aqueous solubility but decreased biological activities and stability. Their low antioxidant activity was correlated with a decreased ph...
Acetone-butanol-ethanol (ABE) fermentation has a high potential for industrial application in lig... more Acetone-butanol-ethanol (ABE) fermentation has a high potential for industrial application in lignocellulose-based biorefinery. Nevertheless, strong end-product inhibition is a severe limitation of this process, which can be partially solved by in situ product recovery techniques, such as extractive fermentation. In this work, the hydrolyzed cellulose from organosolv pre-treated beech wood (Fagus sylvatica L.) was fermented by Clostridium beijerinckii CECT 508 and compared with synthetic P2 medium in conventional and extractive fermentation processes. The conventional control fermentation yielded 9.12 g l−1 of ABE with C. beijerinckii CECT 508, while ABE overproduction of 40% was observed with cellulose hydrolysate as a substrate. After extractive batch fermentation based on 2-butyl-1-octanol as extractant, the final ABE titer was doubled in the P2 control medium over the conventional batch, with respective increases of 50, 190, and 140% for butanol (B), acetone (A) and ethanol (E)....
The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction wi... more The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction with excellent antioxidant properties.
Abstract Background Rutin is a common dietary flavonoid which has received great attention in lit... more Abstract Background Rutin is a common dietary flavonoid which has received great attention in literature, due to their pharmacological properties, including antimicrobial, anti-inflammatory, anticancer, antidiabetic, inter alia . Over 860 products containing rutin are currently marketed in the US. The major disadvantage associated with rutin is its constrained bioavailability, mainly caused by its low aqueous solubility, poor stability and limited membrane permeability. Scope and approach The aim of this contribution is to give an overview of the current methods (conventional and innovative) for the extraction, identification and purification of rutin. Furthermore, recent findings regarding its pharmacological activities and the different approaches to increase rutin solubility in both aqueous and lipid phases will be discussed. Key findings and conclusions Current trends on extraction process have been focused on the discovery and design of green and sustainable extraction techniques to optimize the recovery of rutin. Despite the bioactivity expressed in different in vitro systems, its biological effects in vivo are limited by the poor bioavailability of the flavonoid. The utilization of delivery systems for rutin or its enzymatic or chemical transformation towards highly soluble derivatives have the potential to improve rutin bioavailability, as well as its stability and/or specific biological properties. These novel rutin formulations may bring this promising flavonoid to the forefront of nutraceuticals for the prevention and/or treatment of various chronic human diseases.
ABSTRACT A three-level Box-Behken factorial design combined with the response surface methodology... more ABSTRACT A three-level Box-Behken factorial design combined with the response surface methodology (RSM) was applied as a tool to study the laccase-catalyzed removal of three estrogenic compounds: estrone (E1), estradiol (E2) and ethinylestradiol (EE2), in a continuous enzymatic membrane reactor (EMR). Three main factors affecting the treatment efficiency were considered: enzyme activity, hydraulic residence time (HRT) and oxygenation rate. As expected, laccase activity and HRT showed great effects and, interestingly, it was demonstrated the relevance of the oxygen in the improvement of oxidation reaction kinetics through the rise of the oxygen dissolved above saturation levels. When considering elimination rates as response, optimal conditions were: 1,000 U/L of laccase, 1 h HRT and 60 mgO2/(L•h) of oxygenation rate, predicting 2.82-3.24 mg eliminated/(L•h), which meant 71-81% of oxidation. These optimum conditions were successfully validated, and a 75% of estrogenicity reduction was achieved. On the other hand, only 100 U/L were found as optimal to maximize the efficacy of the enzyme: E1 was oxidized by 0.06 mg/(L•h•U), although the removal of estrogenicity decreased to 60%. The methodology was also applied to maximize the reduction of estrogenic activity: the highest values assayed (1,000 U/L, HRT 4 h and 60 mgO2/(L•h)) provided a 99% of detoxification.
Continuous production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an... more Continuous production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an Expanded-Bed Bioreactor was successfully achieved for more than 30 days of operation. The stability in continuous production was attained because of two modifications in the operational policy: an adequate feed control and the supply of oxygen in a pulsing mode. Feed control allowed to stablish an equilibrium between primary and secondary metabolism, thus avoiding metabolic stress. The use of pulsation had two beneficial effects: 1) Increase of stability by avoiding bed compactation during operation performance; 2) Control of mycelial pellet size. The bioreactor operated without oxygen pulsation did not maintain enzyme production longer than 14 days.
Environmental Science and Pollution Research, Oct 21, 2015
Enzymes immobilization is a useful way to allow enzyme reuse and increase their stability. A high... more Enzymes immobilization is a useful way to allow enzyme reuse and increase their stability. A high redox potential laccase from Trametes versicolor (TvL) and a low redox potential, but commercially available low-cost laccase from Myceliophthora thermophila (MtL), were successfully immobilized and co-immobilized onto fumed silica nanoparticles (fsNP). Enzyme loads of 1.78 ± 0.07, 0.69 ± 0.03, and 1.10 ± 0.01 U/mg fsNP were attained for the optimal doses of TvL, MtL, and co-immobilized laccases, respectively. In general, the laccase-fsNP conjugates showed a higher resistance against an acidic pH value (i.e., pH 3), and a higher storage stability than free enzymes. In addition, immobilized enzymes exhibited a superior long-term stability than free laccases when incubated in a secondary effluent from a municipal wastewater treatment plant (WWTP). For instance, the residual activity after 2 weeks for the co-immobilized laccases and the mixture of free laccases were 40.2 ± 2.5 % and 16.8 ± 1.0 %, respectively. The ability of the laccase-fsNP to remove a mixture of (14)C-bisphenol A (BPA) and (14)C-sodium diclofenac (DCF) from spiked secondary effluents was assessed in batch experiments. The catalytic efficiency was highly dependent on both the microbial source and state of the biocatalyst. The high redox potential TvL in free form attained a four-fold higher percentage of BPA transformation than the free MtL. Compared to free laccases, immobilized enzymes led to much slower rates of BPA transformation. For instance, after 24 h, the percentages of BPA transformation by 1000 U/L of a mixture of free laccases or co-immobilized enzymes were 67.8 ± 5.2 and 27.0 ± 3.9 %, respectively. Nevertheless, the use of 8000 U/L of co-immobilized laccase led to a nearly complete removal of BPA, despite the unfavorable conditions for laccase catalysis (pH ~ 8.4). DCF transformation was not observed for any of the enzymatic systems, showing that this compound is highly recalcitrant toward laccase oxidation under realistic conditions.
The enzyme-mediated polymerization of bioactive phenolic compounds, such as the flavonoid rutin, ... more The enzyme-mediated polymerization of bioactive phenolic compounds, such as the flavonoid rutin, has gained interest due to the enhanced physico-chemical and biological properties of the products, which increases their potential application as a nutraceutical. In this work, the influence of enzyme activity on rutin oligomerization was evaluated in reactions with low (1000 U/L) and high (10,000 U/L) initial laccase activities. For both reactions, high molecular weight oligomer fractions showed better properties compared to lower weight oligomers. Products of the reaction with low laccase activity exhibited thermal stability and antioxidant potential similar to control reaction, but led to higher inhibitory activity of xanthine oxidase and apparent aqueous solubility. Oligomers obtained in the reaction with high laccase activity showed better apparent aqueous solubility but decreased biological activities and stability. Their low antioxidant activity was correlated with a decreased ph...
Acetone-butanol-ethanol (ABE) fermentation has a high potential for industrial application in lig... more Acetone-butanol-ethanol (ABE) fermentation has a high potential for industrial application in lignocellulose-based biorefinery. Nevertheless, strong end-product inhibition is a severe limitation of this process, which can be partially solved by in situ product recovery techniques, such as extractive fermentation. In this work, the hydrolyzed cellulose from organosolv pre-treated beech wood (Fagus sylvatica L.) was fermented by Clostridium beijerinckii CECT 508 and compared with synthetic P2 medium in conventional and extractive fermentation processes. The conventional control fermentation yielded 9.12 g l−1 of ABE with C. beijerinckii CECT 508, while ABE overproduction of 40% was observed with cellulose hydrolysate as a substrate. After extractive batch fermentation based on 2-butyl-1-octanol as extractant, the final ABE titer was doubled in the P2 control medium over the conventional batch, with respective increases of 50, 190, and 140% for butanol (B), acetone (A) and ethanol (E)....
The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction wi... more The oligomerization of esculin by laccase in aqueous medium resulted in a precipitate fraction with excellent antioxidant properties.
Abstract Background Rutin is a common dietary flavonoid which has received great attention in lit... more Abstract Background Rutin is a common dietary flavonoid which has received great attention in literature, due to their pharmacological properties, including antimicrobial, anti-inflammatory, anticancer, antidiabetic, inter alia . Over 860 products containing rutin are currently marketed in the US. The major disadvantage associated with rutin is its constrained bioavailability, mainly caused by its low aqueous solubility, poor stability and limited membrane permeability. Scope and approach The aim of this contribution is to give an overview of the current methods (conventional and innovative) for the extraction, identification and purification of rutin. Furthermore, recent findings regarding its pharmacological activities and the different approaches to increase rutin solubility in both aqueous and lipid phases will be discussed. Key findings and conclusions Current trends on extraction process have been focused on the discovery and design of green and sustainable extraction techniques to optimize the recovery of rutin. Despite the bioactivity expressed in different in vitro systems, its biological effects in vivo are limited by the poor bioavailability of the flavonoid. The utilization of delivery systems for rutin or its enzymatic or chemical transformation towards highly soluble derivatives have the potential to improve rutin bioavailability, as well as its stability and/or specific biological properties. These novel rutin formulations may bring this promising flavonoid to the forefront of nutraceuticals for the prevention and/or treatment of various chronic human diseases.
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