Gotowa bibliografia na temat „GH ENZYMES”

Enzymes classified with the same Enzyme Commission (EC) that are allotted in different glycoside hydrolase (GH) families can display different mechanisms of action and substrate specificities. Therefore, the combination of different enzyme classes may not yield synergism during biomass hydrolysis, as the GH family allocation of the enzymes influences their behavior. As a result, it is important to understand which GH family combinations are compatible to gain knowledge on how to efficiently depolymerize biomass into fermentable sugars. We evaluated GH10 (Xyn10D and XT6) and GH11 (XynA and Xyn2A) β-xylanase performance alone and in combination with various GH family α-l-arabinofuranosidases (GH43 AXH-d and GH51 Abf51A) and α-d-glucuronidases (GH4 Agu4B and GH67 AguA) during xylan depolymerization. No synergistic enhancement in reducing sugar, xylose and glucuronic acid released from beechwood xylan was observed when xylanases were supplemented with either one of the glucuronidases, except between Xyn2A and AguA (1.1-fold reducing sugar increase). However, overall sugar release was significantly improved (≥1.1-fold reducing sugar increase) when xylanases were supplemented with either one of the arabinofuranosidases during wheat arabinoxylan degradation. Synergism appeared to result from the xylanases liberating xylo-oligomers, which are the preferred substrates of the terminal arabinofuranosyl-substituent debranching enzyme, Abf51A, allowing the exolytic β-xylosidase, SXA, to have access to the generated unbranched xylo-oligomers. Here, it was shown that arabinofuranosidases are key enzymes in the efficient saccharification of hetero-xylan into xylose. This study demonstrated that consideration of GH family affiliations of the carbohydrate-active enzymes (CAZymes) used to formulate synergistic enzyme cocktails is crucial for achieving efficient biomass saccharification.

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11TS. Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme–xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11TS. By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11TS.

ABSTRACTSpirochaeta thermophilais a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on theS. thermophilagenome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from theS. thermophilaDSM 6192 genome, all putative β-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, β-glucanase, β-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected inSpirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes ofCytophaga fermentansandMahella australiensis, both of which are phylogenetically very distant fromS. thermophilaand noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.

ABSTRACTRuminococcus albus8 is a fibrolytic ruminal bacterium capable of utilization of various plant cell wall polysaccharides. A bioinformatic analysis of a partial genome sequence ofR. albusrevealed several putative enzymes likely to hydrolyze glucans, including lichenin, a mixed-linkage polysaccharide of glucose linked together in β-1,3 and β-1,4 glycosidic bonds. In the present study, we demonstrate the capacity of four glycoside hydrolases (GHs), derived fromR. albus, to hydrolyze lichenin. Two of the genes encoded GH family 5 enzymes (Ra0453 and Ra2830), one gene encoded a GH family 16 enzyme (Ra0505), and the last gene encoded a GH family 3 enzyme (Ra1595). Each gene was expressed inEscherichia coli, and the recombinant protein was purified to near homogeneity. Upon screening on a wide range of substrates, Ra0453, Ra2830, and Ra0505 displayed different hydrolytic properties, as they released unique product profiles. The Ra1595 protein, predicted to function as a β-glucosidase, preferred cleavage of a nonreducing end glucose when linked by a β-1,3 glycosidic bond to the next glucose residue. The major product of Ra0505 hydrolysis of lichenin was predicted to be a glucotriose that was degraded only by Ra0453 to glucose and cellobiose. Most importantly, the four enzymes functioned synergistically to hydrolyze lichenin to glucose, cellobiose, and cellotriose. This lichenin-degrading enzyme mix should be of utility as an additive to feeds administered to monogastric animals, especially those high in fiber.

β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified an endo-β-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-β-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis. The Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-β-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other β-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

Abstract The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme’s ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.

Abnormal exposure to steroid hormones within a critical developmental period elicits permanent alterations in female reproductive physiology in rodents, but the impact on the female GH axis and the underlying sexual differences in hepatic enzymes have not been described in detail. We have investigated the effect of neonatal androgenization of female mice (achieved by s.c. injection of 100 μg testosterone propionate (TP) on the day of birth: TP females) on the GHRH–somatostatin–GH axis and downstream GH targets, which included female and male predominant liver enzymes and secreted proteins. At 4 months of age, an organizational effect of neonatal testosterone was evidenced on hypothalamic Ghrh mRNA level but not on somatostatin (stt) mRNA level. Ghrh mRNA levels were higher in males than in females, but not in TP females. Increased expression in TP females correlated with increased pituitary GH content and somatotrope population, increased serum and liver IGF-I concentration, and ultimately higher body weight. Murine urinary proteins (MUPs) that were excreted at higher levels in male urine, and whose expression requires pulsatile occupancy of liver GH receptors, were not modified in TP females and neither was liver Mup 1/2/6/8 mRNA expression. Furthermore, a male predominant liver gene (Cyp2d9) was not masculinized in TP females either, whereas two female predominant genes (Cyp2b9 and Cyp2a4) were defeminized. These data support the hypothesis that neonatal steroid exposure contributes to the remodeling of the GH axis and defeminization of hepatic steroid-metabolizing enzymes, which may compromise liver physiology.

Growth hormone (GH) reduces the catabolic side effects of steroid treatment due to its effects on tissue protein synthesis/degradation. Little attention is focused on hepatic amino acid degradation and urea synthesis. Five groups of rats were given 1) placebo, 2) prednisolone, 3) placebo, pair fed to the steroid group, 4) GH, and 5) prednisolone and GH. After 7 days, the in vivo capacity of urea N synthesis (CUNS) was determined by saturating alanine infusion, in parallel with measurements of liver mRNA levels of urea cycle enzymes, N contents of organs, N balance, and hormones. Prednisolone increased CUNS (μmol ⋅ min−1 ⋅ 100 g−1, mean ± SE) from 9.1 ± 1.0 (pair-fed controls) to 13.2 ± 0.8 ( P < 0.05), decreased basal blood α-amino N concentration from 4.2 ± 0.5 to 3.1 ± 0.3 mmol/l ( P < 0.05), increased mRNA levels of the rate- and flux-limiting urea cycle enzymes by 20 and 65%, respectively ( P < 0.05), and decreased muscle N contents and N balance. In contrast, GH decreased CUNS from 6.1 ± 0.9 (free-fed controls) to 4.2 ± 0.5 ( P < 0.05), decreased basal blood α-amino N concentration from 3.8 ± 0.3 to 3.2 ± 0.2, decreased mRNA levels of the rate- and flux-limiting urea cycle enzymes to 60 and 40%, respectively ( P < 0.05), and increased organ N contents and N balance. Coadministration of GH abolished all steroid effects. We found that prednisolone increases the ability of amino N conversion into urea N and urea cycle gene expression. GH had the opposite effects and counteracted the N-wasting side effects of prednisolone.

Abstract The CAZy database is a web-server for sequence-based classification of carbohydrate-active enzymes that has become the worldwide and indispensable tool for scientists engaged in this research field. It was originally created in 1991 as a classification of glycoside hydrolases (GH) and currently, this section of CAZy represents its largest part counting 172 GH families. The present Opinion paper is devoted to the specificity of α-amylase (EC 3.2.1.1) and its occurrence in the CAZy database. Among the 172 defined GH families, four, i.e. GH13, GH57, GH119 and GH126, may be considered as the α-amylase GH families. This view reflects a historical background and traditions widely accepted during the previous decades with respect to the chronology of creating the individual GH families. It obeys the phenomenon that some amylolytic enzymes, which were used to create the individual GH families and were originally known as α-amylases, according to current knowledge from later, more detailed characterization, need not necessarily represent genuine α-amylases. Our Opinion paper was therefore written in an effort to invite the scientific community to think about that with a mind open to changes and to consider the seemingly unambiguous question in the title as one that may not have a simple answer.

Transgenic mice overexpressing growth hormone (GH) have been extensively used to study the chronic effects of elevated serum levels of GH. GH is known to have many acute effects in the liver, but little is known about the chronic effects of GH overexpression on hepatic gene expression. Therefore, we used DNA microarray to compare gene expression in livers from bovine GH (bGH)-transgenic mice and littermates. Hepatic expression of peroxisome proliferator-activated receptor-α (PPARα) and genes involved in fatty acid activation, peroxisomal and mitochondrial β-oxidation, and production of ketone bodies was decreased. In line with this expression profile, bGH-transgenic mice had a reduced ability to form ketone bodies in both the fed and fasted states. Although the bGH mice were hyperinsulinemic, the expression of sterol regulatory element-binding protein (SREBP)-1 and most lipogenic enzymes regulated by SREBP-1 was reduced, indicating that these mice are different from other insulin-resistant models with respect to expression of SREBP-1 and its downstream genes. This study also provides several candidate genes for the well-known association between elevated GH levels and cardiovascular disease, e.g., decreased expression of scavenger receptor class B type I, hepatic lipase, and serum paraoxonase and increased expression of serum amyloid A-3 protein. We conclude that bGH-transgenic mice display marked changes in hepatic genes coding for metabolic enzymes and suggest that GH directly or indirectly regulates many of these hepatic genes via decreased expression of PPARα and SREBP-1.

Les enzymes dégradant les hémicelluloses, comme les xylanases ou les arabinofuranosidases, possèdent un fort potentiel en tant que biocatalyseurs dans des applications industrielles. Ces enzymes sont déjà employées dans des secteurs variés comme l'industrie du papier dans l'étape du blanchiment de la pâte à papier, l'alimentation animale où elles permettent d'accroître la valeur nutritionnelle, l'industrie du vin pour favoriser la libération des arômes, etc. De plus, les hémicellulases apparaissent comme des éléments essentiels dans le futur développement de bioraffineries qui convertiront les déchets agricoles en carburant et autres produits chimiques. Pour améliorer notre compréhension des motifs structuraux qui jouent un rôle dans la fonctionnalité des hémicellulases, nous avons étudié la relation structure/fonction d'enzymes appartenant à deux familles d'hémicellulases. La technique de mutagenèse dirigée a été employée pour explorer différents aspects fonctionnels d'une xylanase GH-11 thermostable (Tx-Xyl), et une analyse cristallographique a été menée sur une arabinofuranosidase GH-51 thermostable (Tx-Abf), ces deux enzymes étant issues du micro-organisme Thermobacillus xylanilyticus. Concernant Tx-Xyl, à la fois sa thermostabilité et un des ses motifs structuraux majeur (le " pouce ") ont été étudiés. Dans le but d'augmenter sa thermostabilité et donc le rendement de dégradation de la biomasse lignocellulosique, une stratégie déjà connue consistant en l'introduction de ponts disulfures a été utilisée. Un mutant affichant un temps de demi-vie à 70°C accru (par un facteur 10) et une plus forte activité spécifique (+ 30%) a été obtenu. Par ailleurs, ce mutant est capable de mieux dégrader les arabinoxylanes constituant le son de blé. Mais de manière surprenante, cette augmentation n'est pas due à la meilleure thermostabilité du mutant. À partir d'études de modélisation moléculaire, des modifications de la composition en acides aminés du pouce de Tx-Xyl sur des positions clés ont contribué à augmenter ou diminuer l'activité catalytique. La suppression du pouce par mutagenèse dirigée a entraîné un changement de spécificité de substrat, et a aboli l'activité enzymatique. En particulier, l'enzyme sans pouce a acquis la faculté de fixer le cellotétraose, sans pour autant que ce ligand ne soit hydrolysé. Enfin, l'étude par cristallographie de Tx-Abf a fourni un modèle structural de cette enzyme à une résolution de 2,1 Å. Cette nouvelle structure, la deuxième dans la famille GH-51, a montré que le domaine catalytique possède une architecture en (b/a)8 et aussi la présence d'un domaine en jelly-roll de fonction inconnue. En comparaison avec l'unique autre structure existant dans cette famille GH-51, Tx-Abf comporte deux ponts disulfures dont l'un est proche du site actif. De plus, la topologie de la crevasse des deux enzymes s'avère différente. La mutagenèse dirigée du résidu Trp248 a révélé son rôle essentiel pendant l'hydrolyse d'arabinoxylo-oligosaccharides de DP3 ou plus
Hemicellulose-degrading enzymes such as xylanases and arabinofuranosidases are potentially useful as biocatalysts for industrial applications. Theses enzymes are already used in numerous sectors such as paper industry for paper pulp whitening, in animal feed preparation to increase nutritional value, in the wine industry to improve bouquet properties etc. In addition, hemicellulases will be key enzymes for the future development of biorefineries which will convert agricultural wastes into transport fuels and other renewable chemicals. To increase understanding of the structural features that are important for the functionality in hemicellulases, we have studied structure/function relationships in enzymes belonging to two hemicellulases families. Directed mutagenesis methods have been used to probe different functional aspects in a thermostable GH-11 endoxylanase (Tx-Xyl), and a crystallographic analysis has been used to study a thermostable GH-51 arabinofuranosidase (Tx-Abf), both enzymes being from Thermobacillus xylanilyticus. Tx-Xyl has been studied with regard to its thermostability and with regard to one of its major structural features, the “thumb”. In order to increase thermostability and hence the usefulness for the degradation of lignocellulosic biomass, an already-tested strategy involving the introduction of disulphide bonds was used. A mutant that exhibits both an increased (10-fold) half-life at 70°C and higher specific activity (30% increase) has been obtained. Gratifyingly, this mutant also displays an increased aptitude for the hydrolysis of arabinoxylans embedded in wheat bran. However, intriguingly, this increase is independent of the increased thermostability. On the basis of a molecular modelling study, modifications of the amino acid composition of Tx-Xyl thumb at key positions lead to increased or decreased catalytic activity. The abolition of the thumb by deletion mutagenesis altered substrate selectivity, and destroyed catalytic activity. In particular, the thumbless enzyme acquired the ability to fix cellotetraose, although this ligand could not be hydrolysed. Finally, a crystallographic study of Tx-Abf has provided a structural model for this enzyme at a resolution of 2. 1 Å. This new structure, the second for GH-51, revealed that the catalytic domain exhibits (b/a)8 architecture and the presence of a jelly-roll domain of unknown function. Compared to the only available structure for GH-51, Tx-Abf contains two putative disulphide bridges, one of which is close to the catalytic apparatus. In addition, the architecture of the catalytic cleft is topologically different to that of the other GH-51 structure. Site-directed mutagenesis of Trp248 has indicated an essential role for this residue in the hydrolysis of arabino-xylo oligosaccharides having a size of DP3 or more

GH enzymes are one of the most important and oldest industrial enzymes. These enzymes are of great significance in present day biotechnology with applications ranging from food, fermentation, pharmaceutical, and detergent, textile and paper industries. GH enzymes are used in detergents to facilitate the removal of starch containing stains. This experimental study aimed at generating recombinant Bacillus subtilis variants expressing significant amount of amylase enzyme. Site directed mutagenesis and site saturation libraries were used to impart mutations in various backbones (wild type strains). This method consists of C-fragment generation and giga PCR (mega primer based PCR). Mutated amylase genes were cloned in Bacillus subtilis and E.coli and transformed colonies were selected based on the presence of zone of clearance. The amylase gene of these clones was amplified by colony PCR and sent for DNA sequencing. The sequence confirmed variants were then fermented in different media for optimization. Expression study of GH enzymes was done using Sodium Dodecyl PolyAcrylamide Gel Electrophoresis (SDS PAGE). Change in the level of expression in different amylase backbones with respect to change in temperature and media volume was also studied. We have used a method for the fast and efficient cloning of GH enzyme genes from various sources by combining the ability of Bacillus subtilis to clone amplify and express the respective genes and of Aspergillus oryzae to efficiently express the heterologous genes. Recombinant protein production was screened in Aspergillus oryzae. Based on the characterization of the enzymes by expression analysis, purification and assay studies, large scale production may be undertaken.