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

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

Interacción entre la reflexión y la generación de formas de lecho

Este artículo presenta un análisis de la evolución temporal de las características hidrodinámicas del oleaje, por medio de la valoración del coeficiente de reflexión, y de las características morfodinámicas del lecho, a través del perfil de fondo. Se ha realizado una serie de 10 ensayos en canal de oleaje bajo las siguientes premisas: reflexión parcial del oleaje originado por un dique vertical poroso, oleaje fuera de la zona de rompientes y transporte del sedimento por fondo. En el análisis experimental se observa que durante la generación y evolución del lecho, se reduce el módulo del coeficiente de reflexión entre un 4% y un 9%, así como que la fase se reduce, a su vez, desplazando la envolvente del oleaje y del perfil de fondo hasta un veinteavo de longitud de onda. Los efectos adversos de este desplazamiento en el frente del dique expuesto al oleaje son por una parte la sobrelevación del oleaje, y por otra, la erosión del lecho a pie de dique

Synthesis and characterization of a lipase-friendly DES based on cholinium dihydrogen phosphate

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGThe main objective of the present work was to synthesize and characterize three novel lipase-friendly deep eutectic solvents (DESs) using cholinium dihydrogen phosphate (N1112OHDHP) as hydrogen bond acceptor and ethylene glycol (EG) and/or glycerol (GL) as hydrogen bond donors in the following proportions: N1112OHDHP:EG (molar ratio 1:2), N1112OHDHP:GL (molar ratio 1:2) and N1112OHDHP:EG:GL (molar ratio 1:1:1). FTIR, 1H NMR, TGA and DSC were employed to shed light on their distinctive structural characteristics. Density, refractive index and dynamic viscosity were also measured at different temperatures, and the experimental data were correlated with several well-known equations. Finally, the suitability of the newly synthesized DESs to be employed in biocatalytic reactions was demonstrated by analysing the activity of a commercial enzyme (Candida antarctica lipase B) after being in contact with them.Agencia Estatal de Investigación | Ref. RTI2018-094702-B-I00Xunta de Galicia | Ref. ED481D-2019/01

Influence of water and ethanol in the physical properties of choline glycinate at several temperatures

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGIn this study, a physical characterization (density (q), refractive index (nD), speed of sound (u) and dynamic viscosity (g)) as a function of temperature (298.15–343.15 K) of choline glycinate (ChGly) and its binary mixtures with water or/and methanol over the entire composition range was performed from 298.15 K to 323.15 K. Several derived properties of the pure ionic liquid and excess molar volume (VE m) changes of refractive index on mixing (DnD), deviations in isentropic compressibility (DjS) and viscosity deviations (Dg) have been calculated using the experimental data in the selected temperature range. These experimental data of ChGly have been positively correlated using polynomial equations and the derived magnitudes were successfully fitted to Redlich-Kister equation. Finally, different models have been implemented for the first time to predict the density of the selected binary mixtures as a function of the temperature.Xunta de Galicia | Ref. ED481D-2019/017Agencia Estatal de Investigación | Ref. RTI2018-094702-B-I0