RESUMO
Wheat bran is an abundant and low valued agricultural feedstock rich in valuable biomolecules as arabinoxylans (AX) and ferulic acid with important functional and biological properties. An integrated bioprocess combining subcritical water extraction (SWE) and enzymatic treatments has been developed for maximised recovery of feruloylated arabinoxylans and oligosaccharides from wheat bran. A minimal enzymatic cocktail was developed combining one xylanase from different glycosyl hydrolase families and a feruloyl esterase. The incorporation of xylanolytic enzymes in the integrated SWE bioprocess increased the AX yields up to 75%, higher than traditional alkaline extraction, and SWE or enzymatic treatment alone. The process isolated AX with tailored molecular structures in terms of substitution, molar mass, and ferulic acid, which can be used for structural biomedical applications, food ingredients and prebiotics. This study demonstrates the use of hydrothermal and enzyme technologies for upcycling agricultural side streams into functional bioproducts, contributing to a circular food system.
Assuntos
Fibras na Dieta , Hidrolases , Humanos , Fibras na Dieta/metabolismo , Ácidos Cumáricos/metabolismo , Xilanos/metabolismo , Endo-1,4-beta-Xilanases/metabolismoRESUMO
Cereal arabinoxylans (AXs) are complex polysaccharides in terms of their pattern of arabinose and ferulic acid substitutions, which influence their properties in structural and nutritional applications. We have evaluated the influence of the molecular structure of three AXs from wheat and rye with distinct substitutions on the activity of ß-xylanases from different glycosyl hydrolase families (GH 5_34, 8, 10 and 11). The arabinose and ferulic acid substitutions influence the accessibility of the xylanases, resulting in specific profiles of arabinoxylan-oligosaccharides (AXOS). The GH10 xylanase from Aspergillus aculeatus (AcXyn10A) and GH11 from Thermomyces lanuginosus (TlXyn11) showed the highest activity, producing larger amounts of small oligosaccharides in shorter time. The GH8 xylanase from Bacillus sp. (BXyn8) produced linear xylooligosaccharides and was most restricted by arabinose substitution, whereas GH5_34 from Gonapodya prolifera (GpXyn5_34) required arabinose substitution and produced longer (A)XOS substituted on the reducing end. The complementary substrate specificity of BXyn8 and GpXyn5_34 revealed how arabinoses were distributed along the xylan backbones. This study demonstrates that AX source and xylanase specificity influence the production of oligosaccharides with specific structures, which in turn impacts the growth of specific bacteria (Bacteroides ovatus and Bifidobacterium adolescentis) and the production of beneficial metabolites (short-chain fatty acids).
RESUMO
Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 (CE15) are involved in microbial degradation of lignocellulosic plant materials. GEs are capable of degrading complex polymers of lignin and hemicellulose cleaving ester bonds between glucuronic acid residues in xylan and lignin alcohols. GEs promote separation of lignin, hemicellulose and cellulose which is crucial for efficient utilization of biomass as an energy source and feedstock for further processing into products or chemicals. Genes encoding GEs are found in both fungi and bacteria, but, so far, bacterial GEs are essentially unexplored, and despite being discovered >10 years ago, only a limited number of GEs have been characterized. The first laboratory scale example of improved xylose and glucuronic acid release by the synergistic action of GE with cellulolytic enzymes was only reported recently (improved C5 sugar and glucuronic acid yields) and, until now, not much is known about their biotechnology potential. In this review, we discuss the diversity, structure and properties of microbial GEs and consider the status of their action on natural substrates and in biological systems in relation to their future industrial use.
Assuntos
Biotecnologia , Esterases , Ácido Glucurônico , Celulose/metabolismo , Proteínas Fúngicas , Lignina/metabolismoRESUMO
The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/ß-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage ß(1â3)/ß(1â4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins.
Assuntos
Proteínas de Bactérias/genética , Evolução Molecular , Glicosídeo Hidrolases/genética , Glicosiltransferases/genética , Proteínas de Plantas/genética , Populus/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Populus/enzimologia , Estrutura Secundária de ProteínaRESUMO
The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 α-1,6- and GH38 α-1,3-mannosidases (SPy1603 and SPy1604), a GH84 ß-hexosaminidase (SPy1600) and a putative GH2 ß-galactosidase (SPy1586), as well as SPy1599, a family GH1 `putative ß-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (ß/α)(8)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a ß-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-ß-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-ß-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).
Assuntos
Proteínas de Bactérias/química , Glucosidases/química , Família Multigênica , Streptococcus pyogenes/enzimologia , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Genoma Bacteriano , Glucosidases/genética , Oligossacarídeos/química , Oligossacarídeos/genética , Streptococcus pyogenes/genética , Relação Estrutura-Atividade , Especificidade por Substrato/genéticaRESUMO
The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin ß-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Genes de Plantas , Glicosiltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/enzimologia , Ativação Enzimática , Ensaios Enzimáticos , Técnicas de Inativação de Genes , Germinação , Glucanos/metabolismo , Glicosiltransferases/genética , Hidrólise , Hipocótilo/enzimologia , Hipocótilo/genética , Hipocótilo/metabolismo , Dados de Sequência Molecular , Pectinas/metabolismo , Filogenia , Pichia/genética , Pichia/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo , Alinhamento de Sequência , Transcriptoma , Xilanos/metabolismoRESUMO
The ability of ß-glucanases to cleave xyloglucans, a family of highly decorated ß-glucans ubiquitous in plant biomass, has traditionally been overlooked in functional biochemical studies. An emerging body of data indicates, however, that a spectrum of xyloglucan specificity resides in diverse glycoside hydrolases from a range of carbohydrate-active enzyme families-including classic "cellulase" families. This chapter outlines a series of enzyme kinetic and product analysis methods to establish degrees of xyloglucan specificity and modes of action of glycosidases emerging from enzyme discovery projects.
Assuntos
Celulase/metabolismo , Ensaios Enzimáticos/métodos , Glucanos/metabolismo , Xilanos/metabolismo , Sequência de Carboidratos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Glucanos/química , Glucanos/isolamento & purificação , Cinética , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Plantas/química , Especificidade por Substrato , Trichoderma/enzimologia , Xilanos/química , Xilanos/isolamento & purificaçãoRESUMO
The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-ß-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.
Assuntos
Proteínas de Bactérias/química , Glucanos/química , Glicosídeo Hidrolases/química , Paenibacillus/enzimologia , Xilanos/química , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage ß-glucan. Isoforms differed in optimum pH (5.0-7.5), in temperature dependence and in acceptor substrate preferences.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Glicosiltransferases/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Estabilidade Enzimática , Expressão Gênica , Glucanos/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/isolamento & purificação , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xilanos/metabolismoAssuntos
Hidrolases de Éster Carboxílico/química , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Escherichia coli/química , Sequência de Carboidratos , Hidrolases de Éster Carboxílico/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Reorganization and degradation of the wall crosslinking and seed storage polysaccharide xyloglucan by glycoside hydrolase family 16 (GH16) endo-transglycosylases and hydrolases are crucial to the growth of the majority of land plants, affecting processes as diverse as germination, morphogenesis, and fruit ripening. A high-resolution, three-dimensional structure of a nasturtium (Tropaeolum majus) endo-xyloglucanase loop mutant, TmNXG1-DeltaYNIIG, with an oligosaccharide product bound in the negative active-site subsites, has been solved by X-ray crystallography. Comparison of this novel complex to that of the strict xyloglucan endo-transglycosylase PttXET16-34 from hybrid aspen (Populus tremula x tremuloides), previously solved with a xylogluco-oligosaccharide bound in the positive subsites, highlighted key protein structures that affect the disparate catalytic activities displayed by these closely related enzymes. Combination of these "partial" active-site complexes through molecular dynamics simulations in water allowed modeling of wild-type TmNXG1, TmNXG1-DeltaYNIIG, and wild-type PttXET16-34 in complex with a xyloglucan octadecasaccharide spanning the entire catalytic cleft. A comprehensive analysis of these full-length complexes underscored the importance of various loops lining the active site. Subtle differences leading to a tighter hydrogen bonding pattern on the negative (glycosyl donor) binding subsites, together with loop flexibility on the positive (glycosyl acceptor) binding subsites appear to favor hydrolysis over transglycosylation in GH16 xyloglucan-active enzymes.
Assuntos
Glucanos/metabolismo , Glicosídeo Hidrolases/química , Glicosiltransferases/química , Nasturtium/enzimologia , Proteínas de Plantas/química , Xilanos/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Glucanos/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Nasturtium/química , Nasturtium/genética , Pichia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Alinhamento de Sequência , Especificidade por Substrato , Triptofano/química , Xilanos/químicaRESUMO
This study focuses on the manufacture and characterization of model surfaces consisting of end-grafted xyloglucan (XG), a naturally occurring polysaccharide, onto a gold substrate. The now well-established XET-technology was utilized for enzymatic incorporation of a thiol moiety at one end of the xyloglucan backbone. This functionalized macromolecule was subsequently top-down grafted to gold, forming a thiol-bonded xyloglucan brushlike layer. The grafting was monitored in situ with QCM-D, and a significant difference in the adsorbed/grafted amount between unmodified xyloglucan and the thiol-functionalized polymer was observed. The grafted surface was demonstrated to be accessible to enzyme digestion using the plant endo-xyloglucanase TmNXG1. The nanotribological properties toward cellulose of the untreated crystal, brush-modified surface, and enzyme-exposed surfaces were compared with a view to understanding the role of xyloglucan in friction reduction. Friction coefficients obtained by the AFM colloidal probe technique using a cellulose functionalized probe on the xyloglucan brush showed an increase of a factor of 2 after the enzyme digestion, and this result is interpreted in terms of surface roughness. Finally, the brush is shown to exhibit binding to cellulose despite its highly oriented nature.
Assuntos
Celulose/química , Glucanos/química , Glicosídeo Hidrolases/química , Ouro/química , Nanopartículas/química , Nanotecnologia/métodos , Xilanos/química , Cristalização , Microscopia de Força Atômica , Sondas Moleculares/química , Quartzo/química , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure-function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen (Populus tremula x Populus tremuloides). Production of the loop deletion variant Tm-NXG1-DeltaYNIIG yielded an enzyme that was structurally similar to Ptt-XET16-34 and had a greatly increased transglycosylation:hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.
Assuntos
Parede Celular/metabolismo , Evolução Molecular , Glicosídeo Hidrolases/química , Proteínas de Plantas/química , Tropaeolum/enzimologia , Catálise , Cromatografia em Gel , Clonagem Molecular , Cristalografia por Raios X , DNA Complementar/metabolismo , Deleção de Genes , Glucanos , Cinética , Dados de Sequência Molecular , Mutagênese , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligossacarídeos/metabolismo , Filogenia , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato , XilanosRESUMO
The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a beta-1,4-linked d-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.