A novel chemoenzymatic glycosylation strategy: application to lysozyme modification

AbstractHen egg lysozyme has been non-specifically glycosylated using a novel two-step strategy. First, a number of sucrose molecules have been chemically bound to the protein surface lysines, then the glycosidic chains have been enzymically lengthened, using a glycosyltransferase. For this task, a fructosyltransferase and a levansucrase have been tested, the latter appearing as the most effective one. In all cases, reactions have been optimised and several degrees of modification have been obtained. Finally, the effects of the modifications on lysozyme hydrophobicity, hydrolytic activity, hydrolysis substrate affinity and thermostability have been assessed

Production of Food Aroma Compounds: Microbial and Enzymatic Methodologies

Posljednjih su godina sve veći zahtjevi za prirodnim proizvodima prehrambene industrije ohrabrili nevjerojatne pokušaje razvoja biotehnoloških procesa u proizvodnji aromatskih spojeva. Ovaj revijalni prikaz donosi nova dostignuća iz tog područja, osobito ističući mogućnosti primjene biljnih stanica i mikrobnih kultura te enzimske tehnologije u proizvodnji velikog broja aromatskih sastojaka hrane. Razmatra se i poboljšanje ekonomske isplativosti proizvodnje uzgojem mikroorganizama na čvrstoj podlozi.Over the past few years, the increasing demand for natural products in the food industry has encouraged remarkable efforts towards the development of biotechnological processes for the production of flavour compounds. The present paper reviews the recent major achievements reported in this field, with a special emphasis on the potential lying in plant cell, microbial cultures and enzyme technology for the production of a wide range of flavours. The use of solid-state fermentation as a means to improve economical feasibility of these processes is also considered

Heavy metals removal from soil/sediments washing effluents via biocompatible aqueous two-phase systems

The generation of high amounts of soil/sediment washing solutions is one of the main concerns faced in ex situ treatment techniques, so in the present research, we have demonstrated the suitability of an organic salt like sodium potassium tartrate to efficiently promote phase segregation in a model soil washing solution containing biodegradable non-ionic-surfactants (Tween 80 and Tergitol 15S9) and heavy metals (copper, zinc, and cadmium), after a preliminary screening of the demixing effect of different inorganic and organic salts like ammonium nitrate, ammonium acetate and sodium potassium tartrate. The aqueous two-phase systems were characterized in depth at room temperature by defining the immiscibility region and correlating the experimental data with three empirical equations, and the results were discussed in the light of a thermodynamic function like the Gibbs free energy of hydration. Then, the applicability to extract the abovementioned metal ions was researched after TLs determination, observing high heavy metals extraction percentage (over 80 % in some cases). The extraction process was included in the soil washing process at real scale and it was simulated by means of the software SuperPro Designer, that makes up a valuable tool to ease the analysis of the technical viability of the proposed strategy.Universidade de Vigo/CISU

Designing biodegradable aqueous biphasic systems for the selective separation of enzymes

In this work, the salting-out strength of reline eutectic mixture in aqueous solution of the non-ionic surfactant Triton X-102 has been demonstrated at temperatures between 298.15 and 323.15 K as a preliminary step to be employed in downstream operations of a biotechnological process. The phase diagrams data of reline-based Aqueous Two Phase Systems (ATPS) have been correlated using empirical models based on exponential and polynomial equations. The tie-lines have been empirically ascertained through physical properties characterization and Othmer-Tobias equation was proposed to correlate those data. After having studied the effect of the proposed reline eutectic mixture on two model enzymes (protease and lipase), the performance of this ATPS for selectively separating them was evaluated for three different feed compositions on the same tie-line, demonstrating their pertinence to segregate the lipase to one of the phases (yield about 95 %) and the protease to the other phase (yield about 98 %). These promising results are interesting for application in biotechnological reactions where proteases have been proved to be deleterious for lipases activity, as the biocompatibility of the proposed platform does not involve drastic negative effects on any of the enzymes.Universidade de Vigo/CISU

The role of deep eutectic solvents in catalysis. A vision on their contribution to homogeneous, heterogeneous and electrocatalytic processes

Deep eutectic solvents (DESs) are a group of eco-friendly, low-cost and tuneable solvents that have gained significant attention in recent years due to their remarkable properties including high solubility and conductivity, low toxicity and easy recovery. DESs have also demonstrated outstanding catalytic activity, especially as a green alternative to traditional solvents and catalysts in several chemical reactions including organic synthesis, biomass conversion and pharmaceutical manufacturing, among others. In this review, we explore the potential of DESs as co-solvents in reaction media or catalysts in a diversity of organic reactions, focusing on both homogeneous and heterogeneous catalysis and electrocatalysis during the last quinquenium. The review article is organised in three main sections: homogeneous catalysis, heterogeneous catalysis and electrocatalysis. In each section, the most significant and recent research regarding versatile types of organic synthesis reactions is presented and discussed. Furthermore, this review offers insights into the future perspectives of DESs in organic catalysis.Universidade de Vigo/CISU

Design of novel aqueous two-phase systems to be coupled in biological remediation processes

The ability of a biodegradable Deep Eutectic Solvent (DES) reline to induce phase segregation in aqueous solutions of polymeric non-ionic surfactants (Tween 20 and Tween 80) has been demonstrated as a preliminary step to be employed for assisting a biological degradation process of a model contaminant. The binodal curves for systems composed of (surfactants + reline + Water) have been determined at several temperatures, and the experimental data have been correlated using well-known equations. The equilibrium data have been analysed in the light of the operating temperature and the hydrophobicity of surfactants. Tie-lines of these systems have been determined, together with useful parameters such as tie-line length and tie-line slopes. Othmer-Tobias and Bancroft equations have been applied to provide an adequate description of the equilibrium data. Finally, the application of this new approach for the remediation of aqueous polluted effluent has proven to be highly efficient, with extraction percentages exceeding 99% for two model persistent contaminants (Acid Black 48 and Crystal Violet). Consequently, this ATPS is proposed as an extra-stage to enhance the remediation percentagesachieved through biological methods. Furthermore, the process has been simulated using commercial software.Universidade de Vigo/CISU

Extracting extremophilic lipases from aqueous streams by using biocompatible ionic liquids

Financiado para publicación en acceso aberto:Universidade de Vigo/CISUGIn this work, biocompatible ionic liquids based on aminoacids were employed as extractants to separate extremolipases from aqueous streams. First, the influence of aminoacid and dipeptide-based ionic liquids (cholinium glycinate, ChGly, and cholinium glycylglycinate, ChGlygly) on the lipolytic activity of a commercial lipase from Candida antarctica (CaLB) and in-house synthesized extremophilic lipases from Thermus thermophilus HB27 (TtHB27L) and Halomonas sp. LM1C (HL) was investigated. The combination of thermophilic enzyme with ChGly turned out to be the optimum combination for maximizing the biocatalytic performance, clearly improving the levels attained when water was exclusively employed as solvent and also surpassing the activity levels provided for the commercial enzyme CaLB. The salting out capacity of ChGly in aqueous solutions of biodegradable surfactants Tergitol 15S7 and Tergitol 15S9 was discussed, recording immiscibility areas almost covering all the ternary diagrams. The aqueous biphasic systems were experimentally characterized by determining both tie-lines and solubility curves at several temperatures and the data was modelled with relevant equations like Merchuk, Othmer-Tobias and Bancroft ones, as they are the most common ones to describe this kind of equilibrium data. So, ChGly was applied to extract thermophilic and commercial lipases from aqueous solutions at 313.15 K, achieving very high extraction levels (about 100 %) for TtHB27L, which clearly surpasses the maximum extraction values observed for the commercial enzyme (about 80 %). Finally, the process was simulated at real scale through SuperPro Designer v.8.5 for the production of 385 Kg/year of extremolipaseXunta de Galicia | Ref. ED481D-2019/017Ministerio de Ciencia, Innovación y Universidades | Ref. RTI2018-094702-B-I0

Creating a new biocatalytic complex with extremolipases and biocompatible ionic liquids for improved transesterification reactions

The ongoing energy crisis has spurred increased research into sustainable and more competitive methods for producing biofuels, including biodiesel. In this context, the focus of the current study is to underscore the viability of investing in a novel biocatalytic complex. This complex incorporates extremophilic lipases and biocompatible ionic liquids with the aim of achieving exceptionally high conversions in transesterification reactions without generating glycerol. Through a meticulous screening process encompassing various amino acid and dipeptide-based ionic liquids from the ammonium family, cholinium glycinate turned out to be the optimal choice. This selection was driven not only by its enhanced compatibility with a commercially available Candida antarctica lipase B (CaLB) but also with extremophilic enzymes synthesized in-house, derived from halophilic (Halomonas spLM1C) and thermophilic (Thermus thermophilus HB27) strains. Following rigorous testing of both free and immobilized enzymes, the ideal concentration of the ionic liquid in transesterification reactions was determined to be 1% relative to the sunflower oil content. Comparative analysis of conversion rates between immobilized thermophilic lipase and immobilized CaLB revealed the efficacy of the proposed approach. Maximum conversions were found to increase by 20%, with specific conversion rates soaring by approximately 180% when utilizing the immobilized thermophilic lipase. In conclusion, this research ushers in new prospects for advancing the competitiveness of biocatalytic solutions in glycerol-free transesterification reactions, underscoring its potential to revolutionize the landscape of sustainable energy production.Agencia Estatal de Investigación | Ref. RTI2018-094702-B-I00Xunta de Galicia | Ref. ED481D-2019/017Universidade de Vigo/CISU

Choline dihydrogen phosphate-based deep eutectic solvent: a suitable bioplatform for lipase extraction

In this work, we have demonstrated the suitability of a novel Deep Eutectic Solvent (DES) composed of cholinium dihydrogen phosphate and ethylene glycol at a molar ratio of 1:2 (ChDHP:EG) to extract Candida antarctica lipase B from aqueous solutions of non-ionic surfactants Triton X-100 and Tween 80. The binodal curves were firstly explored at temperatures between 298.15 and 333.15 K and the experimental data were correlated by means of empirical models based on exponential and polynomial equations. The tie-lines have been ascertained by means of density and refractive indices measurement, and Othmer-Tobias, Bancroft and Setschenow equations were proposed to correlate these data. The Tween 80-based Aqueous Biphasic System was demonstrated to avoid any deleterious effect on the lipase biocatalytic activity, and more than 80% of the enzyme activity was retained on the DES-rich phase, which proves the suitability of these platforms to be employed in downstream stages in lipase production processes.Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGXunta de Galicia | Ref. ED481D-2019/017Xunta de Galicia | Ref. ED431F 2016/007Agencia Estatal de Investigación | Ref. RTI2018-094702-B-I0

Physico-chemical characterization of methanolic mixtures of cholinium dihydrogen phosphate-based DES

The biocompatibility of three deep eutectic solvents based on cholinium dihydrogen phosphate for their use in lipase-catalyzed reactions was recently demonstrated, so the possible application as cosolvents with methanol in transesterification processes demands an exhaustive characterization of the physical properties. Thus, in this work binary and ternary deep eutectic solvents composed of the abovementioned salt as hydrogen bond acceptor and ethyleneglycol and/or glycerol as hydrogen bond donors were synthesized and mixed with methanol. The density, refractive index, electrical conductivity and dynamic viscosity were determined between 293.15 K and 323.15 K. The experimental data were correlated with different well-known equations and derived magnitudes like excess molar volumes, changes of refractive index on mixing and viscosity deviations were inferred in order to get an in-depth characterization of the studied mixtures with cholinium dihydrogen phosphate-based DES. The most intriguing data observed is the great peak measured for ionic conductivity at very high methanol concentration, which is difficult to explain even with the most recent theoretical models.Agencia Estatal de Investigación | Ref. RTI2018-094702-B-I00Xunta de Galicia | Ref. ED481D-2019/017Universidade de Vigo/CISU