Oxidación de proteínas y lípidos en cerebro de cobayos durante la exposición a las grandes alturas (4540 m)

Se evaluó el efecto del tiempo de exposición a las grandes alturas sobre la oxidación de proteínas y lípidos del tejido cerebral de cobayos nativos del nivel del mar trasladados a las grandes alturas (Morococha, 4540 m), y sacrificados los días 1, 3, 7 y 14 después de su arribo. Se determinó los niveles medios de cuerpos carbonílicos (CC), malondialdehído (MDA) e hidroperóxidos lipídicos (LOOH) como marcadores de la oxidación de proteínas y lípidos respectivamente; así como, las actividades de las enzimas antioxidantes superóxido dismutasa (SOD), catalasa (CAT) y glutation peroxidasa (GPx) y fosfolipasa A2 (FLA2) como mediadora de la peroxidación lipídica. Se encontró niveles de CC, LOOH y MDA incrementados al primer día; CC disminuyó por debajo del control al tercer día, LOOH mantuvo la tendencia a disminuir y MDA mantuvo sus niveles altos. Las actividades de las enzimas antioxidantes: GPx y CAT incrementaron desde el primer día; la actividad de SOD aumentó hasta el tercer día disminuyendo posteriormente; la actividad de FLA2 aumentó hasta el tercer día. Los resultados indican que la exposición por diferentes tiempos a las grandes alturas influye directamente en el proceso de oxidación de proteínas y lípidos. La disminución de los niveles de CC podría deberse a la activación del sistema proteolítico, en especial de las proteasas dependientes de Ca+2 como las calpaínas o del sistema proteasomal, las cuales degradarían las proteínas dañadas por las EROs. La exposición a la altura influye además en la actividad de las enzimas antioxidantes, especialmente en GPx, que juega un rol importante en la detoxificación de LOOH, lo que explicaría la tendencia a disminuir al final del tiempo de estudio.-- It was determined the effect of high-altitude exposition time (Morococha - 4540 m) on protein and lipid oxidation from brain of level-sea native guinea pig for different times (1,3,7 and 14 days). It was measured the level of carbonyl groups (CC), malondialdehyde (MDA) and lipids hydroperoxydes (LOOH) as protein and lipid oxidation markers respectively. Also, the activity of antioxidants enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and phospholipase A2 (PLA2) a mediator of lipid peroxidation were evaluated. The results showed an increase of the CC, LOOH and MDA levels during the first day; CC decreased below the control levels by the third day, LOOH level had a decrease trend in time and MDA kept its higher levels. The activity of both antioxidant enzymes GPx and CAT, increased since the first day. Moreover, the activity of SOD showed an increase up to third day followed by a decrease; the activity of PLA2 increased up to the third day. The dates recorded indicated that the expositions at altitudes for different times affect directly the oxidative process of both protein and lipid. The decrease on the CC level could be caused by the activation of the proteolytic system, especially the activation of calcium-dependent proteases as calpains or the proteasomal system which could degrade damaged proteins by EROs. The exposition of altitude might affect the activity of antioxidant enzymes, especially GPx, which could play an important role in the detoxification of LOOH.Tesi

Synergistic action of thermophilic pectinases for pectin bioconversion into D-galacturonic acid

Large amounts of pectin-rich biomass are generated worldwide yearly, which can be hydrolysed by pectinases to obtain bio-based chemical building blocks such as D-galacturonic acid (GalA). The aim of this work was to investigate thermophilic pectinases and explore their synergistic application in the bioconversion of pectic substrates into GalA. Two exo-polygalacturonases (exo-PGs) from Thermotoga maritima (TMA01) and Bacillus licheniformis (BLI04) and two pectin methylesterases (PMEs) from Bacillus licheniformis (BLI09) and Streptomyces ambofaciens (SAM10) were cloned and expressed in Escherichia coli BL21 (DE3), purified and fully characterised. These pectinases exhibited optimum activity at temperatures above 50 °C and good stability at high temperature (40-90 °C) for up to 24 h. Exo-PGs preferred non-methylated substrates, suggesting that previous pectin demethylation by PMEs was necessary to achieve an efficient pectin monomerisation into GalA. Synergistic activity between PMEs and exo-PGs was tested using pectin from apple, citrus and sugar beet. GalA was obtained from apple and citrus pectin in a concentration of up to 2.5 mM after 4 h reaction at 50 °C, through the combined action of BLI09 PME with either TMA01 or BLI04 exo-PGs. Overall, this work contributes to expand the knowledge of pectinases from thermophiles and provides further insights into their application in the initial valorisation of sustainable pectin-rich biomass feedstocks

Inmovilización de transaminasas y amonio liasas y su aplicación en síntesis de compuestos aminados

El objetivo del presente trabajo de tesis es establecer métodos biocatalíticos para la síntesis de los aminoácidos L-fenilalanina, L-aspartato, β−aminobutirato y de las aminas aromáticas 1-feniletilamina y 3-amino-1-fenilbutano, usando biocatalizadores con actividad transaminasa y amonio liasa inmovilizados por técnicas de formación de enlaces covalentes (en el caso de enzimas) y por atrapamiento (tanto para enzimas como para células). Se establecieron dos métodos enzimáticos para la síntesis del aminoácido aromático esencial L-fenilalanina (Phe). El primer método de síntesis se realizó usando como biocatalizador L-aspartato transaminasa (13T) de corazón porcino inmovilizado. Se optimizaron los métodos de inmovilización de 13T en los soportes Eupergit® C, MANA-agarosa y LentiKats®. Los biocatalizadores inmovilizados resultantes se emplearon en la síntesis de Phe. La inmovilización en Eupergit® C y LentiKats® permitió mejorar la estabilidad del enzima 13T, así como alcanzar rendimientos de reacción de síntesis de Phe superiores al 70%. Además se estableció un método de síntesis multienzimática "one-pot", para lo que se acoplaron las reacciones catalizadas por los enzimas aspartasa (AspB) y transaminasa (AT). Se determinó la compatibilidad de los enzimas en las condiciones de reacción (pH 7,5 y 37°C) y se establecieron las concentraciones óptimas de los sustratos (0,15 M de fumarato, 0,3 M de NH4Cl y 0,1 M de fenilpiruvato) y de los enzimas (0,3 U de AspB/mL y 2 U de AT/mL). En estas condiciones, se alcanzó un rendimiento global de reacción de 80%. Para la síntesis de L-aspartato (Asp) se utilizó el enzima L-aspartato amonio liasa o aspartasa (AspB) de Bacillus sp. YM55-1 parcialmente purificado. El enzima se inmovilizó en los soportes Eupergit® C, MANA-agarosa y LentiKats®. Los biocatalizadores inmovilizados se emplearon en la síntesis de altas concentraciones de Asp (≥ 60 g/L). Además, el enzima inmovilizado se reutilizó eficientemente en la síntesis del aminoácido, manteniendo alrededor del 90% de su actividad inicial al cabo de 5 ciclos de reacción en todos los casos. La síntesis de las aminas aromáticas 1-feniletilamina (FEA) y 3-amino-1-fenilbutano (AFB) se llevó a cabo utilizando biocatalizadores (células y/o enzima parcialmente purificado) con actividad ω−transaminasa (ω−TA). Se estableció un método de permeabilización de las membranas de células ω−TA utilizando bromuro de cetrimonio (CTAB). Las células ω−TA se inmovilizaron en el soporte LentiKats® con un 100% de retención de su actividad catalítica. El enzima ω−TA se inmovilizó en varios soportes, obteniéndose los mejores resultados en Eupergit® CM y LentiKats®. La inmovilización en LentiKats®, tanto de células (no permeabilizadas y permeabilizadas) como del enzima, permitió la reutilización de los catalizadores hasta en 5 y 10 ciclos de síntesis de AFB, manteniendo alrededor del 80 y 70% de la actividad inicial, respectivamente. Por último, se comprobó que el enzima aspartasa mutado (AspB-C6) a partir de AspB, cataliza la reacción de aminación regioselectiva del ácido crotónico para producir β−aminobutirato. Este enzima se inmovilizó en los soportes Eupergit® C y MANA-agarosa según los métodos de inmovilización establecidos para el enzima AspB en los mismos soportes. Se obtuvieron rendimientos de inmovilización similares pero las actividades retenidas fueron significativamente inferiores para AspB-C6 que para AspB.The objective of this thesis is to establish biocatalytic methods for the synthesis of the amino acids L-aspartate, L-phenylalanine and β−aminobutyrate, as well as the aromatic amines 1-phenylethylamine and 3-amine-1-phenylbutane, by using biocatalysts with transaminase and ammonia-lyase activity immobilized by covalent attachment techniques (for enzymes) and entrapment (for enzymes and cells). Two methods for the synthesis of the essential aromatic amino acid L-phenylalanine (Phe) were established. The first method was carried out by using L-aspartate transaminase (AAT) from porcine heart as biocatalyst. The immobilization of the enzyme AAT on Eupergit® C, MANA-agarose and LentiKats® supports was optimized, and the three immobilized enzymatic derivatives were used for the synthesis of Phe. AAT immobilized on Eupergit® C and LentiKats® allowed improving the stability of the enzyme as well as reaching reaction yields of Phe over 70%. Moreover, a multi-enzymatic one-pot method for the synthesis of Phe was established by coupling of two consecutive enzymatic reactions catalyzed by aspartase (AspB) and transaminase (AT). The compatibility of both enzymes under the reaction conditions (pH 7,5 and 37°C) was shown; and, the optimum concentration of substrates (0,15 M fumarate, 0,3 M NH4Cl and 0,1 M phenylpyruvate) and enzymes (0,3 U of AspB/mL and 2 U of AT/mL) were established. In these conditions, the global reaction yield was 80%. L-aspartate was synthesized by using the enzyme L-aspartate ammonia-lyase or aspartase (AspB) from Bacillus sp. YM55-1. The enzyme was immobilized on Eupergit® C, MANA-agarose and LentiKats® supports. The immobilized biocatalysts were used for the synthesis of highly concentrated Asp (≥ 60 g/L). Furthermore, the immobilized biocatalysts were efficiently reused in 5 cycles of Asp synthesis, maintaining over 90% of activity and reaching over 90% of conversion in all the cases. The synthesis of the aromatic amine 3-amine-phenylbutane (APB) was carried out by means of cells with ω−transaminase (ω−TA) activity as well as the partially purified enzyme ω−TA. A permeabilization method of the cell membranes with cetrimonium bromide (CTAB) was performed. Cells were immobilized in LentiKats® with 100% retention of the catalytic activity. The enzyme ω−TA was immobilized on several supports, and the best results were obtained with Eupergit® CM and LentiKats® supports. The cells (non-permeabilized and permeabilized) and the enzyme immobilized in LentiKats®, allowed reusing the biocatalysts up to 5 and 10 cycles of synthesis of APB, maintaining around 80 and 70% of the initial activity respectively. Finally, it was shown that the mutated aspartase (AspB-C6) from AspB, catalyzes the regioselective amination of crotonic acid to yield β−aminobutyrate. This enzyme was immobilized on Eupergit® C and MANA-agarose supports, according to the immobilization methods established for AspB on the same supports. The immobilization yields were similar; however the retained activities were lower than those obtained for AspB

Potential of sugar beet vinasse as a feedstock for biocatalyst production within an integrated biorefinery context

BACKGROUNDThis work explores the feasibility of vinasse as an inexpensive feedstock for industrial biocatalyst production within the context of an integrated sugar beet biorefinery. As an exemplar, production of CV2025 ω‐Transaminase (ω‐TAm) in Escherichia coli BL21 was studied.RESULTSCharacterisation of vinasse showed that it comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 cell growth and CV2025 ω‐TAm production were feasible in cultures using 17% to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The d‐galactose present in vinasse facilitated auto‐induction of the pQR801 plasmid enabling CV2025 ω‐TAm expression without addition of expensive Isopropyl‐β‐d‐thiogalactopyranoside (IPTG). Assessment of different vinasse pre‐processing options confirmed simple dilution of the vinasse was sufficient to reduce the concentration of polyphenols to below inhibitory levels. Optimisation experiments, carried out using a controlled, 24‐well microbioreactor platform, showed supplementation of diluted vinasse medium with 10 g L−1 yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3‐fold in specific growth rate, maximum biomass concentration, CV2025 ω‐TAm volumetric and specific activity, respectively. Investigation into the metabolic preferences of E. coli BL21 when grown in vinasse showed a preference for D‐mannitol utilisation before simultaneous metabolism of glycerol, d‐xylitol, d‐dulcitol and acetate. Scale‐up of optimised conditions for batch CV2025 ω‐TAm production to a 7.5 L stirred tank reactor (STR) was demonstrated based on matched volumetric mass transfer coefficient (kLa). The results showed good comparability with respect to cell growth, substrate consumption and CV2025 ω‐TAm production representing over a 700‐fold volumetric scale translation. Further enhancements in CV2025 ω‐TAm production were possible in the STR when operated at higher kLa values.CONCLUSIONThis work describes the promising application of vinasse for production of microbial enzymes and insights into carbon source utilisation in complex feedstocks. Exploitation of vinasse as a fermentation feedstock could be further extended to other processes involving different microorganisms and target enzymes

Continuous enzymatic hydrolysis of sugar beet pectin and l-arabinose recovery within an integrated biorefinery

Sugar beet pulp (SBP) fractionated by steam explosion, released sugar beet pectin (SB-pectin) which was selectively hydrolysed using a novel α-l-arabinofuranosidase (AF), yielding monomeric l-arabinose (Ara) and a galacturonic acid rich backbone (GABB). AF was immobilised on an epoxy-functionalised resin with 70% overall immobilisation yield. Pretreatment of SB-pectin, to remove coloured compounds, improved the stability of the immobilised AF, allowing its reutilisation for up to 10 reaction cycles in a stirred tank reactor. Continuous hydrolysis of SB-pectin was subsequently performed using a packed bed reactor (PBR) with immobilised AF. Reactor performance was evaluated using a Design of Experiment approach. Pretreated SB-pectin hydrolysis was run for 7 consecutive days maintaining 73% of PBR performance. Continuous separation of Ara from GABB was achieved by tangential flow ultrafiltration with 92% Ara recovery. These results demonstrate the feasibility of establishing a continuous bioprocess to obtain Ara from the inexpensive SBP biomass

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

Transketolase (TK) is a fundamentally important enzyme in industrial biocatalysis which carries out a stereospecific carbon–carbon bond formation, and is widely used in the synthesis of prochiral ketones. This study describes the biochemical and molecular characterisation of a novel and unusual hyperthermophilic TK from Thermotoga maritima DSM3109 (TKtmar). TKtmar has a low protein sequence homology compared to the already described TKs, with key amino acid residues in the active site highly conserved. TKtmar has a very high optimum temperature (>90 °C) and shows pronounced stability at high temperature (e.g. t1/2 99 and 9.3 h at 50 and 80 °C, respectively) and in presence of organic solvents commonly used in industry (DMSO, acetonitrile and methanol). Substrate screening showed activity towards several monosaccharides and aliphatic aldehydes. In addition, for the first time, TK specificity towards uronic acids was achieved with TKtmar catalysing the efficient conversion of D-galacturonic acid and lithium hydroxypyruvate into 7-keto-octuronic acid, a very rare C8 uronic acid, in high yields (98%, 49 mM)

The Discovery of Imine Reductases and their Utilisation for the Synthesis of Tetrahydroisoquinolines

Imine reductases (IREDs) are NADPH-dependent enzymes with significant biocatalytic potential for the synthesis of primary, secondary, and tertiary chiral amines. Their applications include the reduction of cyclic imines and the reductive amination of prochiral ketones. In this study, twenty-nine novel IREDs were revealed through genome mining. Imine reductase activities were screened at pH 7 and 9 and in presence of either NADPH or NAD

Multienzyme one-pot cascades incorporating methyltransferases for the strategic diversification of tetrahydroisoquinoline alkaloids

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological activities. Routes to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and can offer better stereoselectivities than traditional synthetic methods. S-Adenosylmethionine (SAM)-dependent methyltransferases are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe the use of methyltransferases in vitro in multi-enzyme cascades, including for the generation of SAM in situ. Up to seven enzymes were used for the regioselective diversification of natural and non-natural THIQs on an enzymatic preparative scale. Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs. An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule

Multienzyme One‐Pot Cascades Incorporating Methyltransferases for the Strategic Diversification of Tetrahydroisoquinoline Alkaloids

The tetrahydroisoquinoline (THIQ) ring system is present in a large variety of structurally diverse natural products exhibiting a wide range of biological  activities. Routes  to mimic the biosynthetic pathways to such alkaloids, by building cascade reactions in vitro, represents a successful strategy and offers better stereoselectivities than traditional synthetic methods.  (S)-Adenosylmethionine (SAM)  dependent methyltransferases   are crucial in the biosynthesis and diversification of THIQs; however, their application is often limited in vitro by the high cost of SAM and low substrate scope. In this study, we describe  the use  of methyltransferases in vitro in multi-enzyme cascades,  including for the generation of SAM   in situ . Up to seven enzymes  were used  for the regioselective diversification of natural and non-natural THIQs on  an enzymatic  preparative scale.  Regioselectivites of the methyltransferases were dependent on the group at C-1 and presence of fluorine in the THIQs.   An interesting dual activity was also discovered for the catechol methyltransferases used, which were found to be able to regioselectively methylate two different catechols in a single molecule

Centrifugal partition chromatography in a biorefinery context: Separation of monosaccharides from hydrolysed sugar beet pulp

A critical step in the bioprocessing of sustainable biomass feedstocks, such as sugar beet pulp (SBP), is the isolation of the component sugars from the hydrolysed polysaccharides. This facilitates their subsequent conversion into higher value chemicals and pharmaceutical intermediates. Separation methodologies such as centrifugal partition chromatography (CPC) offer an alternative to traditional resin-based chromatographic techniques for multicomponent sugar separations. Highly polar two-phase systems containing ethanol and aqueous ammonium sulphate are examined here for the separation of monosaccharides present in hydrolysed SBP pectin: l-rhamnose, l-arabinose, d-galactose and d-galacturonic acid. Dimethyl sulfoxide (DMSO) was selected as an effective phase system modifier improving monosaccharide separation. The best phase system identified was ethanol:DMSO:aqueous ammonium sulphate (300 g L−1) (0.8:0.1:1.8, v:v:v) which enabled separation of the SBP monosaccharides by CPC (200 mL column) in ascending mode (upper phase as mobile phase) with a mobile phase flow rate of 8 mL min−1. A mixture containing all four monosaccharides (1.08 g total sugars) in the proportions found in hydrolysed SBP was separated into three main fractions; a pure l-rhamnose fraction (>90%), a mixed l-arabinose/d-galactose fraction and a pure d-galacturonic acid fraction (>90%). The separation took less than 2 h demonstrating that CPC is a promising technique for the separation of these sugars with potential for application within an integrated, whole crop biorefinery