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1.
Carbohydr Polym ; 338: 122168, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-38763718

RESUMEN

Enzymatic functionalization of oligosaccharides is a useful and environmentally friendly way to expand their structural chemical space and access to a wider range of applications in the health, food, feed, cosmetics and other sectors. In this work, we first tested the laccase/TEMPO system to generate oxidized forms of cellobiose and methyl ß-D-cellobiose, and obtained high yields of novel anionic disaccharides (>60 %) at pH 6.0. Laccase/TEMPO system was then applied to a mix of cellooligosaccharides and to pure D-cellopentaose. The occurrence of carbonyl and carboxyl groups in the oxidation products was shown by LC-HRMS, MALDI-TOF and reductive amination of the carbonyl groups was attempted with p-toluidine a low molar mass amine to form the Schiff base, then reduced by 2-picoline borane to generate a more stable amine bond. The new grafted products were characterized by LC-HRMS, LC-UV-MS/MS and covalent grafting was evidenced. Next, the same procedure was adopted to successfully graft a dye, the rhodamine 123, larger in size than toluidine. This two-step chemo-enzymatic approach, never reported before, for functionalization of oligosaccharides, offers attractive opportunities to anionic cellooligosaccharides and derived glucoconjugates of interest for biomedical or neutraceutical applications. It also paves the way for more environmentally-friendly cellulose fabric staining procedures.


Asunto(s)
Aminas , Lacasa , Oligosacáridos , Oligosacáridos/química , Aminas/química , Lacasa/química , Lacasa/metabolismo , Óxidos N-Cíclicos/química , Oxidación-Reducción , Celobiosa/química , Bases de Schiff/química
2.
Anal Chem ; 94(4): 2279-2287, 2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-35049286

RESUMEN

Carbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerous biological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by the anomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing in demand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic and chemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power (Rp) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD-MS methods for the precise structural characterization of complex glycans.


Asunto(s)
Polisacáridos , Espectrometría de Masas en Tándem , Carbohidratos , Isomerismo , Oligosacáridos/química , Polisacáridos/química
3.
Sci Rep ; 11(1): 20294, 2021 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-34645865

RESUMEN

Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active [Formula: see text]-transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens.


Asunto(s)
Glucosa/análisis , Glucosa/química , Oligosacáridos/química , Polisacáridos/química , Sacarasa/química , Biotecnología , Carbohidratos/química , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Diseño Asistido por Computadora , Enzimas/química , Glicosilación , Haptenos , Hidrolasas/metabolismo , Biología Molecular , Mutación , Antígenos O , Ingeniería de Proteínas/métodos , Shigella flexneri , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem
4.
Curr Opin Chem Biol ; 61: 96-106, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33360622

RESUMEN

An increasing number of transglycosylase-based processes provide access to oligosaccharides or glycoconjugates, some of them reaching performance levels compatible with industrial developments. Nevertheless, the full potential of transglycosylases has not been explored because of the challenges in transforming a glycoside hydrolase into an efficient transglycosylase. Advances in studying enzyme structure/function relationships, screening enzyme activity, and generating synthetic libraries guided by computational protein design or machine learning methods should considerably accelerate the development of these catalysts. The time has now come for researchers to uncover their possibilities and learn how to design and precisely refine their activity to respond more rapidly to the growing demand for well-defined glycosidic structures.


Asunto(s)
Glicósido Hidrolasas/metabolismo , Tecnología Química Verde , Glicósidos/química , Oligosacáridos/química
5.
Microb Genom ; 6(10)2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32667876

RESUMEN

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target ß-1,2-, ß-1,3- and ß-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known ß-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.


Asunto(s)
Bacterias/enzimología , Microbioma Gastrointestinal/genética , Glicósido Hidrolasas/genética , Manósidos/metabolismo , Fosforilasas/genética , Secuencia de Aminoácidos , Animales , Bacterias/genética , Bacterias/metabolismo , Secuencia de Bases , Bovinos , Humanos , Ratones , Oligosacáridos/metabolismo , Fosforilasas/metabolismo , Análisis de Secuencia de ADN , Porcinos
6.
J Biol Chem ; 295(28): 9474-9489, 2020 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-32409580

RESUMEN

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.


Asunto(s)
Proteínas Bacterianas/química , Glucanos/química , Glicosiltransferasas/química , Leuconostoc/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Glucanos/biosíntesis , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Dominios Proteicos
7.
Appl Biochem Biotechnol ; 187(2): 583-611, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30009326

RESUMEN

The discharge of industrial effluent creates environmental problems around the world and so necessitates the need for the economically expensive and sometimes technically problematic treatment of the wastewater. Laccases have enormous potential for the oxidative bioremediation of toxic xenobiotic compounds using only molecular oxygen as the sole cofactor for their reaction, and their application is regarded as environmentally friendly. Due to the low substrate specificity of laccases, they can oxidize a variety of substrates. Moreover, by using appropriate mediators, laccases can degrade a wide range of substrates, including those with structural complexity. Thus, laccases are an attractive alternative for wastewater treatment. Marine environments are rich in microorganisms that are exposed to extreme conditions, such as salinity, temperature, and pressure. Laccases from these microorganisms potentially have suitable properties that might be adaptive to bioremediation processes. This review provides the latest information on laccases from marine environments, their sources, biochemical properties, media composition for laccase production, and their applications in the bioremediation of industrial waste, especially focusing on dye decolorization.


Asunto(s)
Organismos Acuáticos/enzimología , Lacasa/química , Aguas Residuales/química , Contaminantes del Agua/química , Purificación del Agua/métodos , Biodegradación Ambiental
8.
Biochim Biophys Acta Bioenerg ; 1859(2): 69-77, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-28842179

RESUMEN

The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_ΔDA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H+/H2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.


Asunto(s)
Clostridium acetobutylicum/enzimología , Hidrogenasas/química , Sustitución de Aminoácidos , Proteínas Bacterianas , Clostridium acetobutylicum/genética , Hidrogenasas/genética , Mutación Missense , Dominios Proteicos
9.
Bioresour Technol ; 125: 267-74, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23026343

RESUMEN

The lcc1 gene coding for the laccase from Trametes versicolor DSM11269 was cloned into the genome of Yarrowia lipolytica using either single or multiple integration sites. The levels of the recombinant laccase activity secreted in the culture media were 0.25 and 1 U ml(-1) for single and multiple integrations, respectively. The strain with a single integration was successfully used to express variant libraries which were screened on ABTS substrate. The strain encoding the double mutant L185P/Q214K (rM4A) showed a sixfold enhancement in secreted enzyme activity. The catalytic efficiency of the purified rM-4A laccase was respectively increased 2.4- and 2.8-fold towards ABTS and 2,6-dimethoxyphenol, compared to the rWT. Culture supernatants containing either rWT or rM-4A catalyzed the almost complete decolorization of an Amaranth solution (70 nMs(-1)). Taken together, our results open new perspectives for the use of Y. lipolytica as a molecular evolution platform to engineer laccases with improved properties.


Asunto(s)
Clonación Molecular/métodos , Lacasa/biosíntesis , Lacasa/química , Ingeniería de Proteínas/métodos , Trametes/fisiología , Yarrowia/fisiología , Catálisis , Activación Enzimática , Estabilidad de Enzimas
10.
J Biol Chem ; 287(9): 6642-54, 2012 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-22210773

RESUMEN

Amylosucrases are sucrose-utilizing α-transglucosidases that naturally catalyze the synthesis of α-glucans, linked exclusively through α1,4-linkages. Side products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes. Here, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis (DgAS). The three-dimensional structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying the DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea in complex with turanose. In the amylosucrase from N. polysaccharea (NpAS), key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanoid tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS. Our findings help to understand the inter-relationships between amylosucrase structure, flexibility, function, and stability and provide new insight for amylosucrase design.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Deinococcus/enzimología , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Sacarosa/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Deinococcus/genética , Dimerización , Disacáridos/química , Disacáridos/metabolismo , Estabilidad de Enzimas , Fructosa/química , Fructosa/metabolismo , Glucosa/metabolismo , Glucosiltransferasas/genética , Calor , Isomerismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Sacarosa/química
11.
Chem Commun (Camb) ; 48(9): 1314-6, 2012 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-22158825

RESUMEN

Direct encapsulation of esterase or lipase fused with the silica-precipitating R5 peptide from Cylindrotheca fusiformis in silica particles afforded high yields of active entrapped protein. The hydrolytic activity of both enzymes against p-nitrophenyl butyrate was similarly affected by encapsulation and the enantioselectivity of the esterase was both improved and inverted.


Asunto(s)
Diatomeas/enzimología , Enzimas Inmovilizadas/metabolismo , Esterasas/metabolismo , Lipasa/metabolismo , Péptidos/química , Dióxido de Silicio/química , Secuencia de Aminoácidos , Materiales Biomiméticos/química , Biomimética , Precipitación Química , Diatomeas/química , Activación Enzimática , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Esterasas/química , Lipasa/química , Datos de Secuencia Molecular
12.
Chembiochem ; 10(17): 2760-71, 2009 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-19816890

RESUMEN

Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2-substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild-type BCL could be improved by chemical engineering of the substrate's molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)-2-chloro ethyl 2-bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15-fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.


Asunto(s)
Sitios de Unión , Lipasa/química , Ingeniería de Proteínas/métodos , Acetatos/química , Dominio Catalítico , Lipasa/genética , Lipasa/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estereoisomerismo , Especificidad por Sustrato
13.
Proteins ; 77(3): 509-23, 2009 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-19475702

RESUMEN

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so-called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position-restrained MD simulations. Our conclusions indicate that the sole mobility of alpha9 helix side-chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by alpha5 helix movement. The role of selected alpha5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase.


Asunto(s)
Bioquímica/métodos , Burkholderia cepacia/enzimología , Lipasa/química , Biología Computacional/métodos , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Fosfatos/química , Conformación Proteica , Proteínas/química , Solventes/química , Factores de Tiempo
14.
Chembiochem ; 9(8): 1308-17, 2008 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-18418817

RESUMEN

A novel approach based on efficient path-planning algorithms was applied to investigate the influence of substrate access on Burkholderia cepacia lipase enantioselectivity. The system studied was the transesterification of 2-substituted racemic acid derivatives catalysed by B. cepacia lipase. In silico data provided by this approach showed a fair qualitative agreement with experimental results, and hence the potential of this computational method for fast screening of racemates. In addition, a collision detector algorithm used during the pathway searches enabled the rapid identification of amino acid residues hindering the displacement of substrates along the deep, narrow active-site pocket of B. cepacia lipase and thus provided valuable information to guide the molecular engineering of lipase enantioselectivity.


Asunto(s)
Burkholderia cepacia/enzimología , Lipasa/química , Lipasa/metabolismo , Biología Computacional , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Especificidad por Sustrato
15.
J Biomol Screen ; 13(1): 72-9, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18227227

RESUMEN

The screening of variant libraries of recombinant Burkholderia cepacia ATCC21808 lipase generated in Escherichia coli is limited by expression difficulties that are mainly due to the formation of inclusion bodies. To circumvent these difficulties and provide an efficient small-scale screening protocol, the gene encoding the lipase from B. cepacia was expressed in various expression vectors. With the pFLAG-ATS-Lip-Hp construct, expression of up to 6807 U/L of culture was possible in Erlenmeyer flasks. The production protocol was miniaturized in 96 deep-well plates, yielding 1300 U/L of lipase in fusion with the FLAG tag. With this protocol, the activity was determined in less than 10 min for a full plate, with a coefficient of variance of about 25%. For validation, 18 mutants constructed by site-directed mutagenesis on position Valine 266 were screened. Nice variations of activity were detected and found to be in agreement with those obtained in Erlenmeyer flask cultures. The protocol enabled the identification of 5 mutants showing enhanced activity toward para-nitrophenyl butyrate.


Asunto(s)
Burkholderia cepacia/enzimología , Burkholderia cepacia/genética , Evaluación Preclínica de Medicamentos/métodos , Lipasa/biosíntesis , Lipasa/genética , Secuencia de Bases , Cartilla de ADN/genética , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Vectores Genéticos , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética
16.
Biotechnol Bioeng ; 82(6): 664-9, 2003 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-12673765

RESUMEN

An economically pertinent process for the lipase-catalyzed synthesis of amides was developed. A continuous plug flow reactor was used. The model reaction was the production of oleamide, a lubricant and anti-slip agent, via direct Candida antarctica lipase B-catalyzed amidation of oleic acid with ammonia. Of all solvents tested, 2-methyl-2-butanol was found to respond optimally to the demands formulated in our specifications. A continuous conversion of oleic acid into oleamide of 85% was obtained. A productivity of 4.5 tons oleamide per kg of enzyme per year was calculated, indicating a contribution of enzyme to the product price of only 4%. The volumetric productivity, 100 g. L(-1). h(-1), is 4 to 100 times higher than in literature procedures. A simple crystallization procedure leads to 99% purity.


Asunto(s)
Amoníaco , Butanoles/química , Lipasa/química , Modelos Químicos , Ácido Oléico/química , Ácidos Oléicos/síntesis química , Pentanoles , Amidas/síntesis química , Catálisis , Simulación por Computador , Enzimas Inmovilizadas , Europa (Continente) , Proteínas Fúngicas , Ácidos Oléicos/economía , Proyectos Piloto , Solventes/química , Estados Unidos
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