Enzymatic synthesis and molecular modelling studies of rhamnose esters using lipase from Pseudomonas stutzeri

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterifica-tion of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 ºC, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99–92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4–C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12–C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observe

Biocatalyzed synthesis of glycostructures with anti-infective activity

Molecules containing carbohydrate moieties play essential roles in fighting a variety of bacterial and viral infections. Consequently, the design of new carbohydrate-containing drugs or vaccines has attracted great attention in recent years as means to target several infectious diseases. Conventional methods to produce these compounds face numerous challenges because their current production technology is based on chemical synthesis, which often requires several steps and uses environmentally unfriendly reactants, contaminant solvents, and inefficient protocols. The search for sustainable processes such as the use of biocatalysts and eco-friendly solvents is of vital importance. Therefore, their use in a variety of reactions leading to the production of pharmaceuticals has increased exponentially in the last years, fueled by recent advances in protein engineering, enzyme directed evolution, combinatorial biosynthesis, immobilization techniques, and flow biocatalysis. In glycochemistry and glycobiology, enzymes belonging to the families of glycosidases, glycosyltransferases (Gtfs), lipases, and, in the case of nucleoside and nucleotide analogues, also nucleoside phosphorylases (NPs) are the preferred choices as catalysts. In this Account, on the basis of our expertise, we will discuss the recent biocatalytic and sustainable approaches that have been employed to synthesize carbohydrate-based drugs, ranging from antiviral nucleosides and nucleotides to antibiotics with antibacterial activity and glycoconjugates such as neoglycoproteins (glycovaccines, GCVs) and glycodendrimers that are considered as very promising tools against viral and bacterial infections. In the first section, we will report the use of NPs and N-deoxyribosyltransferases for the development of transglycosylation processes aimed at the synthesis of nucleoside analogues with antiviral activity. The use of deoxyribonucleoside kinases and hydrolases for the modification of the sugar moiety of nucleosides has been widely investigated. Next, we will describe the results obtained using enzymes for the chemoenzymatic synthesis of glycoconjugates such as GCVs and glycodendrimers with antibacterial and antiviral activity. In this context, the search for efficient enzymatic syntheses represents an excellent strategy to produce structure-defined antigenic or immunogenic oligosaccharide analogues with high purity. Lipases, glycosidases, and Gtfs have been used for their preparation. Interestingly, many authors have proposed the use Gtfs originating from the biosynthesis of natural glycosylated antibiotics such as glycopeptides, macrolides, and aminoglycosides. These have been used in the chemoenzymatic semisynthesis of novel antibiotic derivatives by modification of the sugar moiety linked to their complex scaffold. These contributions will be described in the last section of this review because of their relevance in the fight against the spreading phenomenon of antibiotic resistance. In this context, the pioneering in vivo synthesis of novel derivatives obtained by genetic manipulation of producer strains (combinatorial biosynthesis) will be shortly described as well. All of these strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can contribute to the sustainable development of complex glycan-based therapies and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and selectivity. The use of enzymatic synthesis is becoming a reality in the pharmaceutical industry and in drug discovery to rapidly afford collections of new antibacterial or antiviral molecules with improved specificity and better metabolic stability

Solvents derived from glycerol modify classical regioselectivity in the enzymatic synthesis of disaccharides with Biolacta β-galactosidase

Green solvents made from glycerol change the classical regioselectivity of Biolacta No 5 β-galactosidase, from β(1→4) to β(1→6) linkages when a 2 M concentration was used. In order to explain these results, the non-proteic compounds present in the Biolacta preparation were separated by precipitation with ammonium sulfate and the remaining protein extract was used to set reactions with appropriate organic solvents to find that the regioselectivity towards the β(1→6) isomer is retained. According to proteomic analysis, a 98% homology between Streptococcus pneumoniae and Biolacta β-galactosidase preparation was found. With these data, molecular modelling was done which predicts a tridimensional interaction in the enzyme active site with the donor (GlcNAc) and the water-solvent mixture which explains this phenomenon. © 2011 The Royal Society of Chemistry.This work was supported by two research projects of the Spanish MICINN (Ministerio de Ciencia e Innovación de España) CTQ2009-11801 and CTQ2008-05138, and one European project (FP-62003-NMP-SMF-3, proposal 011774-2).Peer Reviewe

A Novel Lipase from Streptomyces exfoliatus DSMZ 41693 for Biotechnological Applications

Genome mining of Streptomyces exfoliatus DSMZ 41693 has allowed us to identify four different lipase-encoding sequences, and one of them (SeLipC) has been successfully cloned and extracellularly expressed using Rhodococcus sp. T104 as a host. SeLipC was purified by one-step hydrophobic interaction chromatography. The enzyme is a monomeric protein of 27.6 kDa, which belongs to subfamily I.7 of lipolytic enzymes according to its phylogenetic analysis and biochemical characterization. The purified enzyme shows the highest activity at 60 °C and an optimum pH of 8.5, whereas thermal stability is significantly improved when protein concentration is increased, as confirmed by thermal deactivation kinetics, circular dichroism, and differential scanning calorimetry. Enzyme hydrolytic activity using p-nitrophenyl palmitate (pNPP) as substrate can be modulated by different water-miscible organic cosolvents, detergents, and metal ions. Likewise, kinetic parameters for pNPP are: KM = 49.6 µM, kcat = 57 s−1, and kcat/KM = 1.15 × 106 s−1·M−1. SeLipC is also able to hydrolyze olive oil and degrade several polyester-type polymers such as poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), and poly(ε-caprolactone) (PCL). Moreover, SeLipC can catalyze the synthesis of different sugar fatty acid esters by transesterification using vinyl laurate as an acyl donor, demonstrating its interest in different biotechnological applications.This research was funded by the Ministerio de Ciencia, Innovación y Universidades of Spain, grants RTI2018-096037B-I00, TED2021-131462B-I00, TED2021-130430B-C21, and PDC2022-133817-I00.Peer reviewe

Highly efficient and regioselective enzymatic synthesis of ß-(1-3) galactosides in biosolvents

A green synthesis for ß-(1-3) galactosyldisaccharides that combines the use of a biodegradable biocatalyst, aqueous solutions, and solvent recycling (renewable and derived from biomass) has been developed. The use of biomass-derived solvents allows good catalytic activity in the synthesis of Gal-ß-d-(1-3)GlcNAc and Gal-ß-d-(1-)3GalNAc (99% and 95% yields respectively) with ß-Gal-3-NTag ß-galactosidase, preventing hydrolytic activity and with full regioselectivity. This represents a considerable improvement over the use of an aqueous buffer or conventional organic solvents. Furthermore, reaction scaling up and biosolvent recycling are feasible without losing catalytic action. In order to understand the role of these green solvents in the enzyme's synthetic behaviour, different structural studies were performed (fluorescence and molecular modelling) in the presence of some selected biosolvents to conclude that the presence of green biosolvents in the reaction media modifies the enzyme's tertiary structure allowing better substrate disposition in the active site, most probably due to solvation effects, explaining the behaviour observed.Peer Reviewe

Síntesis de nuevos compuestos fructoconjugados mediante el empleo de la ß-fructofuranosidasa de Schwanniomyces occidentalis

Trabajo presentado en la 2ª ed. del congreso nacional "Jornadas Españolas de Biocatálisis" (JEB) organizado por la Sociedad Española de Biotecnología en el Campus de El Cristo de la Universidad de Oviedo (Asturias, España) durante los días 25 al 26 de junio de 2018.Los procesos sintéticos mediados por enzimas están adquiriendo hoy en día cada vez mayor importancia en la industria, especialmente para la obtención de moléculas altamente específicas de manera poco costosa y respetuosa con el medio ambiente. En este contexto, las levaduras no convencionales ofrecen una verdadera oportunidad para descubrir nuevas enzimas alternativas que permitan desempeñar acciones catalíticas inéditas con múltiples posibles aplicaciones. Recientemente, el ascomiceto Schwanniomyces occidentalis ha suscitado interés biotecnológico por disponer de un amplio sistema glicosilhidrolítico extracelular de elevada operatividad y robustez. Su enzima β-fructofuranosidasa (EC 3.2.1.26) no sólo se ha mostrado capaz de escindir eficientemente enlaces fructosídicos β-(2→1) ó β-(2→6) presentes en diversos sacáridos o heterósidos (actividad hidrolítica); sino que también tiene el potencial para transfructosilar con distinto grado de regioselectividad ciertos sustratos aceptores a partir de donadores no activados como la sacarosa (actividad transferente). El estudio en detalle de esta habilidad catalítica ha permitido conocer con mayor exactitud la especificidad aceptora de esta enzima sobre una gran cantidad de azúcares y glicitoles. Las reacciones con dos de los glicitoles que mostraron mayor rendimiento, eritritol y manitol, fueron escaladas y los productos obtenidos purificados y caracterizados. Asimismo, se han puesto en práctica distintas estrategias para aumentar la tasa de transfructosilación de los aceptores positivos, como son la optimización de las concentraciones de sustrato, la variación de los tiempos de reacción y el empleo de biodisolventes, tanto derivados de biomasa como líquidos iónicos. Todo ello está generando como resultado la formación biocatalítica de nuevos compuestos fructoconjugados de posible interés bioactivo que serían de lo contrario difíciles de obtener por síntesis química clásica.N

Highly efficient enzymatic synthesis of Galβ-(1→3)-GalNAc and Galβ-(1→3)-GlcNAc in ionic liquids

Ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability, negligible vapour pressure, non-flammability and easy recycling. In this context, this work assesses the catalytic activity of a β-galactosidase from Bacillus circulans ATCC 31382 (β-Gal-3-NTag) in the synthesis of β-(1→3)-galactosides using different ILs. A noticeably increase in activity, retaining total regioselectivity was found in the synthetic behaviour of B. circulans β-galactosidase in ILs as co-solvents and using a 1:5 molar ratio of donor (pNP-β-Gal):acceptor (GlcNAc or GalNac). Yields up to 97% of β-(1→3) with different ILs were found. These reactions take place without noticeable hydrolytic activity and with total regioselectivity, representing a considerable improvement over the use of aqueous buffer or conventional organic solvents. Furthermore, reaction scaling up and IL recovery and recycling are feasible without losing catalytic action. Molecular modelling studies performed predict a three-dimensional interaction at the active centre between the acceptor and the water-IL mixture, which could explain the results obtained. © 2013 Elsevier Ltd. All rights reserved.Peer Reviewe

Screening of strains and recombinant enzymes from Thermus thermophilus for their use in disaccharide synthesis

Cell extracts from ten different strains of Thermus thermophilus have been screened for glycosidase activity. Among these, the sequenced strain HB27 hydrolyzed a wide variety of glycosides and increased six fold its β-glycosidase activity when grown with cellobiose in nutrient-limited media. We selected five genes encoding (putative) glycosidases (TTP0042, TTP0072, TTP0220, TTC0107 and TTP0222) from the genome of this strain, and the corresponding recombinant enzymes were overexpressed and purified. Several transglycosylation reactions using cellobiose-induced HB27 cell extracts and the purified recombinant enzymes were assayed. Biochemical properties and biosynthetic capabilities of the HB27 cell extracts and the TTP0042 enzyme were very similar, suggesting that this enzyme was responsible for most of the β-glycosidase activity detected in the HB27 strain. This was confirmed through the isolation and analysis of a null mutant of its encoding gene. With both, HB27 cell extracts and purified TTP0042 recombinant enzyme, we finally achieved high yields conditions for disaccharide production by transglycosylation with low amounts of self-condensed donor when high concentrations of a 1:5 donor:acceptor molar ratio was used. © 2011 Elsevier B.V. All rights reserved.Ministerio de Ciencia e Innovacion (CTQ2009-11801, BIO2010-18875); Universidad Nacional de Costa Rica; Ministerio de Educacion; Fundacion Ramon ArecesPeer Reviewe

Efficient and selective enzymatic synthesis of N-acetyl-lactosamine in ionic liquid: a rational explanation

Room temperature ionic liquids (ILs) can affect enzyme activity in some enzyme-catalyzed reactions, however the effects of these cosolvents on the enzymes are not clearly understood. Using β-galactosidase from Thermus thermophilus HB27 (TTP0042), we found an important change from the classical regioselectivity of the transglycosylation reaction with this enzyme. The enzyme increases N-acetyl-D-lactosamine synthesis (Galβ[1→4]GlcNAc) when RTILs are used instead of the traditional self-condensated products. To understand the possible effect of these liquids on the synthetic behavior of the enzyme, we performed a molecular interaction study by surface plasmon resonance. The KD value obtained for this interaction could mean that ILs bind to β-galactosidase through non specific interactions characterized by very fast kinetics and millimolar affinity. Then, several reactions were performed, increasing the concentration of the IL. As a result, a dependence on the IL's concentration was found for transglycosylation products. We hypothesize that ILs might induce conformational changes in the enzyme, which would modify the enzymatic activity and regioselectivity. These structural modifications were confirmed in the secondary and tertiary structures of the protein by circular dichroism and fluorescence studies, respectively. Molecular modeling confirms this hypothesis and shows that the enzyme becomes more flexible in an IL–water mixture and that it allows stabilization of the GlcNAc molecule in the active centre of the enzyme, in order to develop a new product according to the original regioselectivity of the reaction.This work has been supported by four research projects, three of the MICINN (‘‘Ministerio de Ciencia e Innovacio´n’’), CTQ2009-11801, BIO2010-18875 and MAT 2008-02542; and one of the Complutense University GR35/10-A-950247 This work has been supported by four research projects, three of the MICINN (‘‘Ministerio de Ciencia e Innovacio´n’’), CTQ2009-11801, BIO2010-18875 and MAT 2008-02542; and one of the Complutense University GR35/10-A-950247. Manuel Sandoval thanks a PhD fellowship granted by Universidad Nacional de Costa Rica.Peer reviewe

Optimised N-acetyl-D-lactosamine synthesis using Thermus thermophilus β-galactosidase in bio-solvents

Synthesis of N-acetyl-D-lactosamine (Gal-β[1→4]GlcNAc, LacNAc) catalyzed by β-galactosidase from Thermus thermophilus (TTP0042) is affected by side reactions that give as result very low yields (about 20%) of LAcNAc when the reaction is performed in buffer. The process is improved (up to 91% of disaccharide yield) when the reaction takes place in the presence of solvents from biomass (bio-solvents) at 2.0 M concentration. Most of the solvents tested increased the LacNAc synthesis and reduced the undesired side reactions. In order to understand the possible effects of these solvents over the enzyme regioselectivity, we developed a conformational study of the enzyme structure in the presence of a selected bio-solvent by circular dichroism and fluorescence. According to this study, we were able to conclude that the presence of bio-solvents in the reaction media modifies the enzyme secondary and tertiary structure and this may be the cause of the regioselectivity changes observed in the transglycosylation reaction. © 2012 Elsevier Ltd. All rights reserved.Ministerio de Ciencia e Innovacion; UCM/ Santander; Universidad Nacional de Costa RicaPeer Reviewe