Integrated biocatalytic platform based on aqueous biphasic systems for the sustainable oligomerization of rutin

Rutin is a known antioxidant compound that displays a broad range of biological activities and health-related benefits but presents a low water solubility that can be overcome by its polymerization. In this work, biocompatible aqueous biphasic systems composed of the ionic liquid cholinium dihydrogen phosphate ([CH][DHph]) and the polymer poly(ethylene glycol) 600 (PEG 600) were investigated as an efficient integrated reaction–separation platform for the laccase-catalyzed oligomerization of rutin. Two different approaches were studied to reuse laccase in several oligorutin production cycles, the main difference between them being the use of monophasic or biphasic regimes during the oligomerization reaction. The use of a biphasic regime in the second approach (heterogeneous reaction medium) allowed the successful reuse of the biocatalyst in three consecutive reaction–separation cycles while achieving noteworthy rutin oligomerization yields (95% in the first cycle, 91% in the second cycle, and 89% in the last cycle). These remarkable results were caused by the combination of the increased solubility of rutin in the PEG-rich phase together with the enhanced catalytic performance of laccase in the [Ch][DHph]-rich phase, alongside with the optimization of the pH of the reaction medium straightly linked to enzyme stability. Finally, a life-cycle assessment was performed to compare this integrated reaction–separation platform to three alternative processes, reinforcing its sustainabilityThis research was supported by the Spanish Government (AEI) through the RTI2018-094482-J-I00 project. This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES. The programme is cofunded by FEDER (UE). A.P.M.T. thanks the FCT for the research contract CEECIND/2020/01867. G.E. thanks the Spanish MICIU for her Ramón y Cajal contract (RYC2018- 024846-I). A.M.-M. thanks the Programa de axudas á etapa predoutoral da Xunta de Galicia (ED481A-2018/023)S

Tecnologías enzimáticas para la eliminación de compuestos de baja solubilidad en agua

La presencia de compuestos recalcitrantes en efluentes, suelos y sedimentos es un problema medioambiental de especial relevancia. Los compuestos de baja solubilidad tales como hidrocarburos aromáticos policíclicos (HAPs) tienen una alta persistencia ya que, debido a sus propiedades físico-químicas, su degradación por las bacterias autóctonas se ve limitada. Con el objetivo de desarrollar un sistema capaz de llevar a cabo la degradación de estos compuestos, se ha planteado la utilización de los hongos ligninolíticos o bien sus enzimas oxidativas. La enzima manganeso peroxidasa (MnP) promueve la oxidación de Mn+2 a Mn+3, el cual actúa como un poderoso agente oxidante que oxida compuestos orgánicos recalcitrantes de forma inespecífica. La degradación de compuestos de baja solubilidad mediante la enzima MnP requiere la adición de un agente que incremente la solubilidad del compuesto a fin de facilitar la acción de la enzima. En este trabajo se plantea la adición de disolventes miscibles e inmiscibles como alternativas paraincrementar la solubilidad de un HAP modelo tal como el antraceno de cara a su degradación enzimática

Formulation of Laccase Nanobiocatalysts Based on Ionic and Covalent Interactions for the Enhanced Oxidation of Phenolic Compounds

Oxidative biocatalysis by laccase arises as a promising alternative in the development of advanced oxidation processes for the removal of xenobiotics. The aim of this work is to develop various types of nanobiocatalysts based on laccase immobilized on different superparamagnetic and non-magnetic nanoparticles to improve the stability of the biocatalysts. Several techniques of enzyme immobilization were evaluated based on ionic exchange and covalent bonding. The highest yields of laccase immobilization were achieved for the covalent laccase nanoconjugates of silica-coated magnetic nanoparticles (2.66 U mg−1 NPs), formed by the covalent attachment of the enzyme between the aldehyde groups of the glutaraldehyde-functionalized nanoparticle and the amino groups of the enzyme. Moreover, its application in the biotransformation of phenol as a model recalcitrant compound was tested at different pH and successfully achieved at pH 6 for 24 h. A sequential batch operation was carried out, with complete recovery of the nanobiocatalyst and minimal deactivation of the enzyme after four cycles of phenol oxidation. The major drawback associated with the use of the nanoparticles relies on the energy consumption required for their production and the use of chemicals, that account for a major contribution in the normalized index of 5.28 × 10−3. The reduction of cyclohexane (used in the synthesis of silica-coated magnetic nanoparticles) led to a significant lower index (3.62 × 10−3); however, the immobilization was negatively affected, which discouraged this alternative

Laccase Activity as an Essential Factor in the Oligomerization of Rutin

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 phenolic content, which could be attributed to the formation of several bonds between rutin molecules

Removal of Estrogenic Compounds from Filtered Secondary Wastewater Effluent in a Continuous Enzymatic Membrane Reactor. Identification of Biotransformation Products

In the present study, a novel and efficient technology based on the use of an oxidative enzyme was developed to perform the continuous removal of estrogenic compounds from polluted wastewaters. A 2 L enzymatic membrane reactor (EMR) was successfully operated for 100 h with minimal requirements of laccase for the transformation of estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)­from both buffer solution and real wastewater (filtered secondary effluent). When the experiments were performed at high and low concentrations of the target compounds, 4 mg/L and 100 μg/L, not only high removal yields (80–100%) but also outstanding reduction of estrogenicity (about 84–95%) were attained. When the EMR was applied for the treatment of municipal wastewaters with real environmental concentrations of the different compounds (0.29–1.52 ng/L), excellent results were also achieved indicating the high efficiency and potential of the enzymatic reactor system. A second goal of this study relied on the identification of the transformation products to elucidate the catalytic mechanism of estrogens’ transformation by laccase. The formation of dimers and trimers of E1, E2, and EE2, as well as the decomposition of E2 into E1 by laccase-catalyzed treatment, has been demonstrated by liquid chromatography atmospheric pressure chemical ionization (LC-APCI) analysis and confirmed by determination of accurate masses through liquid chromatography electrospray time-of-flight mass spectrometry (LC-ESI-TOF). Dimeric products of E2 and EE2 were found even when operating at environmental concentrations. Moreover, the reaction pathways of laccase-catalyzed transformation of E2 were proposed

Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts

6 páginas, 4 figuras, 1 tabla -- PAGS nros. 129-134Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 °C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L-1 was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulansThis work was partially supported by the EU contract “Fungal metalloenzymes oxidizing aromatic compounds of industrial interest” (QLK3-99-590), the Comunidad de Madrid, and the Spanish Commission of Science and Technology (BIO98-610 and BIO99-908)Peer reviewe

Comprehensive investigation of the enzymatic oligomerization of esculin by laccase in ethanol : water mixtures

The enzymatic polymerization of phenolic compounds arouses increasing interest due to the production of derivatives with improved biological activity. The reaction yield, the molecular mass, the structure and the properties of synthesized polymers can be controlled by the reaction conditions such as solvent and type of enzyme and substrate. In this study, the oxidative oligomerization of esculin by laccase from Trametes versicolor was performed in the presence of ethanol, a biocompatible co-solvent for food and nutraceutical applications. The formation of a precipitate was associated with the oligomerization reaction except for the medium with 50% (v/v) ethanol, due to the low reaction yield. The evaluation of antioxidant activity of the monomer and products showed that the pellet fraction from the reaction with esculin at 2 g L−1 in acetate buffer led to the highest activities. The presence of esculin oligomers was confirmed by MALDI-TOF analysis, which identified a repetition unit of 338 Da with a degree of polymerization up to 9 as well as other oligomers, mainly in the pellet fraction, with a repetition unit of 176 Da which are attributed to be esculetin oligomers. Additionally, size exclusion chromatography (SEC) and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the products.This work was financially supported by the Spanish Ministry of Economy and Competitiveness (CTQ2014-58879-JIN). Authors belong to the Galician Competitive Research Group GRC 2013-032 and to the strategic group CRETUS (AGRUP2015/02). All these programmes are co-funded by FEDER. B. G. thanks the Spanish Ministry of Economy and Competitiveness for her postdoctoral fellowship (Grant reference FPDI-2013-17341)S