Zebrafish (Danio rerio) are currently used for various biomedical and environmental studies. Due to the comprehensive ethological and genetical description, but also due to a significant similarity ...to humans, zebrafish are successfully used in modelling some traits associated with human pathology (both psychiatric and others) and determining the effects of environmental factors on behaviour and highly conserved molecular processes. Due to the fact that chronic exposure to stress is a major problem for contemporary society, any description of the phenomenon and finding solutions to minimize its harmful effects are important research directions. Therefore, through this study, we aimed to evaluate and describe some behavioural and oxidative changes that occur following exposure to chronic stress, in an adapted animal model. Twenty zebrafish were acclimatized to laboratory conditions and randomly divided into 2 study groups: a control group and a group that was exposed to a combination of stressors for 10 consecutive days. Following stress exposure, locomotor activity and affective state were assessed. Afterwards, biological samples were collected and used to determine some oxidative status parameters (the enzymatic activity of superoxide dismutase, of glutathione peroxidase, and the amount of malondialdehyde). We obtained significant variations of some behavioural parameters suggesting a significant harmful effect of chronic stress on the locomotor activity. Also, manifestations of anxious-depressive behaviours were observed, as compared to the control group. Biochemical analyses revealed significant changes in the activity of the antioxidant enzymes and increased lipid peroxidation which suggests that exposure to chronic stress could lead to the occurrence of oxidative stress. KEYWORDS: Chronic stress, anxiety, oxidative stress, animal model, zebrafish.
In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains ...with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.
•Use of immobilized antibodies to get the one step immobilization–purification•Use of domains with specific affinity for some ligands to immobilize/purify tagged proteins•Immobilization/purification by using of domains that increase the enzyme affinity for standard supports•Glyoxyl supports and one step immobilization–purification of multimeric proteins•Medium engineering and standard supports for immobilization/purification•Tailor made heterofunctional supports for large protein immobilization/purification
Pseudothermotoga hypogea is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding P. hypogea ...ADH (PhADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other Thermotoga species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of PhADH contains an α/β-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant PhADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant PhADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant PhADH in E. coli significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs.
E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the ...E1 for the Ubl small ubiquitin-like modifier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics.
Enzyme immobilization on inorganic materials is gaining more attention with the potential characteristics of high-surface-area-to-volume ratios, increasing the efficiency of enzyme loading on the ...support. Metal oxide hybrid support was prepared by a wetness impregnation of five metal precursors, including CaO, CuO, MgO, NiO, and ZnO, on Alsub.2Osub.3 and used as a support for the immobilization of Candida rugosa lipase (CRL) by adsorption. Maximum activity recovery (70.6%) and immobilization efficiency (63.2%) were obtained after optimization of five parameters using response surface methodology (RSM) by Box-Behnken design (BBD). The biochemical properties of immobilized CRL showed high thermostability up to 70 °C and a wide range in pH stability (pH 4-10). TGA-DTA and FTIR analysis were conducted, verifying thermo-decomposition of lipase and the presence of an amide bond. FESEM-EDX showed the homogeneous distribution and high dispersion of magnesium and CRL on MgO-Alsub.2Osub.3, while a nitrogen adsorption-desorption study confirmed MgO-Alsub.2Osub.3 as a mesoporous material. CRL/MgO-Alsub.2Osub.3 can be reused for up to 12 cycles and it demonstrated high tolerance in solvents (ethanol, isopropanol, methanol, and tert-butanol) compared to free CRL.
The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these ...water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization may improve enzyme stability. Starting from the stabilization effects that an enzyme may experience by the mere fact of being inside a solid particle, we detail other possibilities to stabilize enzymes: generation of favorable enzyme environments, prevention of enzyme subunit dissociation in multimeric enzymes, generation of more stable enzyme conformations, or enzyme rigidification via multipoint covalent attachment. In this last point, we will discuss the features of an “ideal” immobilization protocol to maximize the intensity of the enzyme-support interactions. The most interesting active groups in the support (glutaraldehyde, epoxide, glyoxyl and vinyl sulfone) will be also presented, discussing their main properties and uses. Some instances in which the number of enzyme-support bonds is not directly related to a higher stabilization will be also presented. Finally, the possibility of coupling site-directed mutagenesis or chemical modification to get a more intense multipoint covalent immobilization will be discussed.
•Enzyme immobilization in a porous structure may protect the enzyme from some inactivating causes•Enzyme immobilization may freeze some stable enzyme conformation•Multi-subunit enzyme immobilization may prevent enzyme subunit dissociation•Enzyme immobilization may enhance enzyme stability by generating special environments•Enzyme multipoint covalent attachment should increase enzyme rigidity
Abstract
Thirty years have elapsed since the emergence of the classification of carbohydrate-active enzymes in sequence-based families that became the CAZy database over 20 years ago, freely ...available for browsing and download at www.cazy.org. In the era of large scale sequencing and high-throughput Biology, it is important to examine the position of this specialist database that is deeply rooted in human curation. The three primary tasks of the CAZy curators are (i) to maintain and update the family classification of this class of enzymes, (ii) to classify sequences newly released by GenBank and the Protein Data Bank and (iii) to capture and present functional information for each family. The CAZy website is updated once a month. Here we briefly summarize the increase in novel families and the annotations conducted during the last 8 years. We present several important changes that facilitate taxonomic navigation, and allow to download the entirety of the annotations. Most importantly we highlight the considerable amount of work that accompanies the analysis and report of biochemical data from the literature.
Skepinone-L was recently reported to be a p38alpha MAP kinase inhibitor with high potency and excellent selectivity invitro and invivo. However, this class of compounds still act as fully ...ATP-competitive TypeI binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first TypeI1 /2 binders for p38alpha MAP kinase. TypeI1 /2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regionsI and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography.
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