RESUMO
As part of the multifaceted strategies developed to shape the common environmental policy, considerable attention is now being paid to assessing the degree of environmental degradation in soil under xenobiotic pressure. Bisphenol A (BPA) has only been marginally investigated in this ecosystem context. Therefore, research was carried out to determine the biochemical properties of soils contaminated with BPA at two levels of contamination: 500 mg and 1000 mg BPA kg-1 d.m. of soil. Reliable biochemical indicators of soil changes, whose activity was determined in the pot experiment conducted, were used: dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, arylsulfatase, and ß-glucosidase. Using the definition of soil health as the ability to promote plant growth, the influence of BPA on the growth and development of Zea mays, a plant used for energy production, was also tested. As well as the biomass of aerial parts and roots, the leaf greenness index (SPAD) of Zea mays was also assessed. A key aspect of the research was to identify those of the six remediating substances-molecular sieve, zeolite, sepiolite, starch, grass compost, and fermented bark-whose use could become common practice in both environmental protection and agriculture. Exposure to BPA revealed the highest sensitivity of dehydrogenases, urease, and acid phosphatase and the lowest sensitivity of alkaline phosphatase and catalase to this phenolic compound. The enzyme response generated a reduction in the biochemical fertility index (BA21) of 64% (500 mg BPA) and 70% (1000 mg BPA kg-1 d.m. of soil). The toxicity of BPA led to a drastic reduction in root biomass and consequently in the aerial parts of Zea mays. Compost and molecular sieve proved to be the most effective in mitigating the negative effect of the xenobiotic on the parameters discussed. The results obtained are the first research step in the search for further substances with bioremediation potential against both soil and plants under BPA pressure.
Assuntos
Fosfatase Ácida , Compostos Benzidrílicos , Fenóis , Poluentes do Solo , Solo , Zea mays , Fenóis/química , Compostos Benzidrílicos/química , Poluentes do Solo/química , Zea mays/química , Solo/química , Fosfatase Ácida/metabolismo , Arilsulfatases/metabolismo , Fosfatase Alcalina/metabolismo , Zeolitas/química , Oxirredutases/metabolismo , Urease/metabolismo , Catalase/metabolismo , Biodegradação Ambiental , Silicatos de Magnésio/química , Amido/química , beta-Glucosidase/metabolismo , Compostagem/métodosRESUMO
Biomass pretreatment for the production of second-generation (2G) ethanol and biochemical products is a challenging process. The present study investigated the synergistic efficiency of purified carboxymethyl cellulase (CMCase), ß-glucosidase, and xylanase from Aspergillus fumigatus JCM 10253 in the hydrolysis of alkaline-pretreated sugarcane bagasse (SCB). The saccharification of pretreated SCB was optimised using a combination of CMCase and ß-glucosidase (C + ß; 1:1) and addition of xylanase (C + ß + xyl; 1:1:1). Independent and dependent variables influencing enzymatic hydrolysis were investigated using response surface methodology (RSM). Hydrolysis using purified CMCase and ß-glucosidase achieved yields of 18.72 mg/mL glucose and 6.98 mg/mL xylose. Incorporation of xylanase in saccharification increased the titres of glucose (22.83 mg/mL) and xylose (9.54 mg/mL). Furthermore, characterisation of SCB biomass by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy respectively confirmed efficient structural disintegration and revealed the degree of crystallinity and spectral characteristics. Therefore, depolymerisation of lignin to produce high-value chemicals is essential for sustainable and competitive biorefinery development.
Assuntos
Aspergillus fumigatus , Biomassa , Celulose , Saccharum , Hidrólise , Aspergillus fumigatus/enzimologia , Celulase/metabolismo , Xilose/metabolismo , beta-Glucosidase/metabolismo , Açúcares/metabolismoRESUMO
To convert ginsenosides Rb1, Rb2, Rb3, and Rc into Rd by a single enzyme, a putative ß-glycosidase (Pxbgl) from the xylan-degrading bacterium Petroclostridium xylanilyticum was identified and used. The kcat/Km value of Pxbgl for Rb3 was 18.18 ± 0.07 mM-1/s, which was significantly higher than those of Pxbgl for other ginsenosides. Pxbgl converted almost all Rb3 to Rd with a productivity of 5884 µM/h, which was 346-fold higher than that of only ß-xylosidase from Thermoascus aurantiacus. The productivity of Rd from the Panax ginseng root and Panax notoginseng leaf was 146 and 995 µM/h, respectively. Mutants N293 K and I447L from site-directed mutagenesis based on bioinformatics analysis showed an increase in specific activity of 29 and 7% toward Rb3, respectively. This is the first report of a ß-glycosidase that can simultaneously remove four different glycosyls at the C-20 position of natural PPD-type ginsenosides and produce Rd as the sole product from P. notoginseng leaf extracts with the highest productivity.
Assuntos
Proteínas de Bactérias , Ginsenosídeos , Panax , Ginsenosídeos/metabolismo , Ginsenosídeos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Panax/química , Panax/genética , Panax/metabolismo , Especificidade por Substrato , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/química , Cinética , beta-Glucosidase/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/química , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Panax notoginseng/química , Panax notoginseng/genética , Panax notoginseng/enzimologia , Panax notoginseng/metabolismoRESUMO
The bovine rumen contains a large consortium of residential microbes that release a variety of digestive enzymes for feed degradation. However, the utilization of these microbial enzymes is still limited because these rumen microorganisms are mostly anaerobes and are thus unculturable. Therefore, we applied a sequence-based metagenomic approach to identify a novel 2,445-bp glycoside hydrolase family 3 ß-glucosidase gene known as BrGH3A from the metagenome of bovine ruminal fluid. BrGH3A ß-glucosidase is a 92-kDa polypeptide composed of 814 amino acid residues. Unlike most glycoside hydrolases in the same family, BrGH3A exhibited a permuted domain arrangement consisting of an (α/ß)6 sandwich domain, a fibronectin type III domain and a (ß/α)8 barrel domain. BrGH3A exhibited greater catalytic efficiency toward laminaribiose than cellobiose. We proposed that BrGH3A is an exo-acting ß-glucosidase from Spirochaetales bacteria that is possibly involved in the intracellular degradation of ß-1,3-/1,4-mixed linkage glucans that are present in grass cell walls. BrGH3A exhibits rich diversity in rumen hydrolytic enzymes and may represent a member of a new clan with a permuted domain topology within the large family.
Assuntos
Rúmen , beta-Glucosidase , Animais , Bovinos , Rúmen/microbiologia , Rúmen/enzimologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , beta-Glucosidase/química , Sequência de Aminoácidos , Filogenia , Domínios Proteicos , MetagenomaRESUMO
BACKGROUND: As promising biomarkers of diabetes, α-glucosidase (α-Glu) and ß-glucosidase (ß-Glu) play a crucial role in the diagnosis and management of diseases. However, there is a scarcity of techniques available for simultaneously and sensitively detecting both enzymes. What's more, most of the approaches for detecting α-Glu and ß-Glu rely on a single-mode readout, which can be affected by multiple factors leading to inaccurate results. Hence, the simultaneous detection of the activity levels of both enzymes in a single sample utilizing multiple-readout sensing approaches is highly attractive. RESULTS: In this work, we constructed a facile sensing platform for the simultaneous determination of α-Glu and ß-Glu by utilizing a luminescent covalent organic framework (COF) as a fluorescent indicator. The enzymatic hydrolysis product common to both enzymes, p-nitrophenol (PNP), was found to affect the fluorometric signal through an inner filter effect on COF, enhance the colorimetric response by intensifying the absorption peak at 400 nm, and induce changes in RGB values when analyzed using a smartphone-based color recognition application. By combining fluorometric/colorimetric measurements with smartphone-assisted RGB mode, we achieved sensitive and accurate quantification of α-Glu and ß-Glu. The limits of detection for α-Glu were determined to be 0.8, 1.22, and 1.85 U/L, respectively. Similarly, the limits of detection for ß-Glu were 0.16, 0.42, and 0.53 U/L, respectively. SIGNIFICANCE: Application of the proposed sensing platform to clinical serum samples revealed significant differences in the two enzymes between healthy people and diabetic patients. Additionally, the proposed sensing method was successfully applied for the screening of α-Glu inhibitors and ß-Glu inhibitors, demonstrating its viability and prospective applications in the clinical management of diabetes as well as the discovery of antidiabetic medications.
Assuntos
Inibidores de Glicosídeo Hidrolases , Estruturas Metalorgânicas , alfa-Glucosidases , beta-Glucosidase , Estruturas Metalorgânicas/química , Humanos , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/química , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/metabolismo , alfa-Glucosidases/metabolismo , alfa-Glucosidases/sangue , Colorimetria/métodos , Limite de Detecção , Nitrofenóis/metabolismo , Nitrofenóis/química , Nitrofenóis/análise , Avaliação Pré-Clínica de Medicamentos , Corantes Fluorescentes/químicaRESUMO
Broussonetine S (9), its C-1' and C-10' stereoisomers, and their corresponding enantiomers have been synthesized from enantiomeric arabinose-derived cyclic nitrones, with cross metathesis (CM), epoxidation and Keck asymmetric allylation as key steps. Glycosidase inhibition assays showed that broussonetine S (9) and its C-10' epimer (10'-epi-9) were nanomolar inhibitors of bovine liver ß-galactosidase and ß-glucosidase; while their C-1' stereoisomers were 10-fold less potent towards these enzymes. The glycosidase inhibition results and molecular docking calculations revealed the importance of the configurations of pyrrolidine core and C-1' hydroxyl for inhibition potency and spectra. Together with the docking calculations we previously reported for α-1-C-alkyl-DAB derivatives, we designed and synthesized a series of 6-C-alkyl-DMDP derivatives with very simple alkyl chains. The inhibition potency of these derivatives was enhanced by increasing the length of the side chain, and maintained at nanomolar scale inhibitions of bovine liver ß-glucosidase and ß-galactosidase after the alkyl groups are longer than eight or ten carbons for the (6R)-C-alkyl-DMDP derivatives and their 6S epimers, respectively. Molecular docking calculations indicated that each series of 6-C-alkyl-DMDP derivatives resides in the same active site of ß-glucosidase or ß-galactosidase with basically similar binding conformations, and their C-6 long alkyl chains extend outwards along the hydrophobic groove with similar orientations. The increasing inhibitions of ß-glucosidase and ß-galactosidase with the number of carbon atoms in the side chains may be explained by improved adaptability of longer alkyl chains in the hydrophobic grooves. In addition, the lower ß-glucosidase and ß-galactosidase inhibitions of (6S)-C-alkyl-DMDP derivatives than their C-6 R stereoisomers can be attributed to the misfolding of their alkyl chains and resulted decreased adaptability in the hydrophobic groove. The work reported herein is valuable for design and development of more potent and selective inhibitors of ß-galactosidase and ß-glucosidase, which have potential in treatment of lysosomal storage diseases. Furthermore, part of the 6-C-alkyl-DMDP derivatives and their enantiomers were also tested as potential anti-cancer agents; all the compounds tested were found with moderate cytotoxic effects on MKN45 cells, which would indicate potential applications of these iminosugars in development of novel anticancer agents.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Simulação de Acoplamento Molecular , beta-Galactosidase , beta-Glucosidase , beta-Galactosidase/antagonistas & inibidores , beta-Galactosidase/metabolismo , Bovinos , Animais , Relação Estrutura-Atividade , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/metabolismo , Estrutura Molecular , Relação Dose-Resposta a Droga , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/químicaRESUMO
Abscisic acid (ABA) plays a crucial role in plant defense mechanisms under adverse environmental conditions, but its metabolism and perception in response to heavy metals are largely unknown. In Pisum sativum exposed to CdCl2, an accumulation of free ABA was detected in leaves at different developmental stages (A, youngest, unexpanded; B1, youngest, fully expanded; B2, mature; C, old), with the highest content found in A and B1 leaves. In turn, the content of ABA conjugates, which was highest in B2 and C leaves under control conditions, increased only in A leaves and decreased in leaves of later developmental stages after Cd treatment. Based on the expression of PsNCED2, PsNCED3 (9-cis-epoxycarotenoid dioxygenase), PsAO3 (aldehyde oxidase) and PsABAUGT1 (ABA-UDP-glucosyltransferase), and the activity of PsAOγ, B2 and C leaves were found to be the main sites of Cd-induced de novo synthesis of ABA from carotenoids and ABA conjugation with glucose. In turn, ß-glucosidase activity and the expression of genes encoding ABA receptors (PsPYL2, PsPYL4, PsPYL8, PsPYL9) suggest that in A and B1 leaves, Cd-induced release of ABA from inactive ABA-glucosyl esters and enhanced ABA perception comes to the forefront when dealing with Cd toxicity. The distinct role of leaves at different developmental stages in defense against the harmful effects of Cd is discussed.
Assuntos
Ácido Abscísico , Cádmio , Regulação da Expressão Gênica de Plantas , Pisum sativum , Folhas de Planta , Proteínas de Plantas , Ácido Abscísico/metabolismo , Pisum sativum/metabolismo , Pisum sativum/efeitos dos fármacos , Pisum sativum/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Cádmio/metabolismo , Cádmio/toxicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Dioxigenases/metabolismo , Dioxigenases/genética , beta-Glucosidase/metabolismo , beta-Glucosidase/genéticaRESUMO
In order to more efficiently utilize the abundant cellulose resources in nature, increase the utilization rate of cellulose in aquaculture, implement precise feeding and save aquaculture costs, we have conducted research on cellulase genes related to the spotted knifejaw (Oplegnathus punctatus). Cellulose, as the most abundant renewable resource, is a cornerstone in the intricate ecological balance of diverse ecosystems. While herbivorous fish are recognized for their utilization of proteins, sugars, and fats, the extent of cellulose utilization by carnivorous and omnivorous fish remains an enigma. Here, through field sampling and behavioural observations, O. punctatus' omnivorous diet has been demonstrated (stomach contents contain approximately several species of algae in the Bacillariophyta (1.12 %), Streptomyces (0.55 %), Chlorophyta (0.35 %), Rhodophyta (0.16 %), and Euglenophyta (0.19 %) phylum). Additionally, the high cellulase activity in the intestine of O. punctatus has been detected first discovery (enzyme activity up to 4800.15 U/g), indicating its ability to digest cellulose. By employing whole-genome scanning and high-throughput sequencing, a single cellulase gene (ß-glucosidase) within the genome of O. punctatus, suggesting the absence of a complete cellulose digestive system. However, microbiological analysis revealed the three crucial role of microorganisms, including Actinobacteria (25.80 %), Bacteroidetes (18.93 %), and Firmicutes phylum (0.82 %), were found to play a crucial role in the decomposition of plant cell walls, thereby facilitating plant material digestion to help the host to complete the process of cellulose digestion. Expression patterns and proteomic analysis of the ß-glucosidase were notably high in the gonads. In situ hybridization confirmed the expression of the ß-glucosidase gene in the intestinal contents and gonads, highlighting its role in supplying energy of gonads. These discoveries shed light on the dietary habits of O. punctatus and its cellulose utilization, offering insights that can inform the development of customized feeding strategies to enhance aquaculture sustainability and minimize resource expenditure.
Assuntos
Peixes , beta-Glucosidase , Animais , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Peixes/genética , Filogenia , Celulose/metabolismo , CarnivoridadeRESUMO
Particulate matter hydrolysis is the bottleneck in anaerobic treatment of municipal wastewater in temperate climates. Low temperatures theoretically slow enzyme-substrate interactions, hindering utilization kinetics, but this remains poorly understood. ß-glucosidase, protease, and lipase activities were evaluated in two pilot-scale upflow anaerobic sludge blanket (UASB) reactors, inoculated with different sludges and later converted to anaerobic membrane bioreactors (AnMBRs). Despite similar methane production and solids hydrolysis rates, significant differences emerged. Specific activity peaked at 37 °C, excluding the predominance of psychrophilic enzymes. Nevertheless, the Michaelis-Menten constant (Km) indicated high enzyme-substrate affinity at the operational temperature of 15-20 °C, notably greater in AnMBRs. It is shown, for the first time, that different seed sludges can equally adapt, as hydrolytic enzymatic affinity to the substrate reached similar values in the two reactors at the operational temperature and identified that membrane ultrafiltration impacted hydrolysis by a favourable enzyme Michaelis-Menten constant.
Assuntos
Reatores Biológicos , Águas Residuárias , Purificação da Água , Hidrólise , Águas Residuárias/química , Anaerobiose , Purificação da Água/métodos , Clima , Lipase/metabolismo , Esgotos , Temperatura , Cinética , Peptídeo Hidrolases/metabolismo , Metano/metabolismo , beta-Glucosidase/metabolismo , Eliminação de Resíduos Líquidos/métodosRESUMO
Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum, Fusarium nygamai, Trichoderma capillare, and Aspergillus calidoustus, are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of ß-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare, SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 µg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus. Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.
Assuntos
Fermentação , Lignina , Olea , Lignina/metabolismo , Olea/microbiologia , Aspergillus/enzimologia , Aspergillus/metabolismo , Celulase/metabolismo , Celulase/biossíntese , Lacase/metabolismo , Lacase/biossíntese , Penicillium/enzimologia , Penicillium/metabolismo , beta-Glucosidase/metabolismo , beta-Glucosidase/biossíntese , Fusarium/enzimologia , Fusarium/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Fungos/enzimologia , Fungos/metabolismo , Marrocos , Proteínas Fúngicas/metabolismoRESUMO
Herein, a novel multifunctional enzyme ß-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of ß-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of ß-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.
Assuntos
Camellia sinensis , Hidrolases de Éster Carboxílico , Chá , beta-Glucosidase , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Camellia sinensis/química , Camellia sinensis/enzimologia , Chá/química , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Odorantes/análiseRESUMO
Beta-glucosidases catalyze the hydrolysis of the glycosidic bonds of cellobiose, producing glucose, which is a rate-limiting step in cellulose biomass degradation. In industrial processes, ß-glucosidases that are tolerant to glucose and stable under harsh industrial reaction conditions are required for efficient cellulose hydrolysis. In this study, we report the molecular cloning, Escherichia coli expression, and functional characterization of a ß-glucosidase from the gene, CelGH3_f17, identified from metagenomics libraries of an Ethiopian soda lake. The CelGH3_f17 gene sequence contains a glycoside hydrolase family 3 catalytic domain (GH3). The heterologous expressed and purified enzyme exhibited optimal activity at 50 °C and pH 8.5. In addition, supplementation of 1 M salt and 300 mM glucose enhanced the ß-glucosidase activity. Most of the metal ions and organic solvents tested did not affect the ß-glucosidase activity. However, Cu2+ and Mn2+ ions, Mercaptoethanol and Triton X-100 reduce the activity of the enzyme. The studied ß-glucosidase enzyme has multiple industrially desirable properties including thermostability, and alkaline, salt, and glucose tolerance.
Assuntos
Biomassa , Lagos , beta-Glucosidase , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , beta-Glucosidase/química , Lagos/microbiologia , Metagenômica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Metagenoma , Clonagem Molecular , Estabilidade Enzimática , Hidrólise , Concentração de Íons de Hidrogênio , Celulose/metabolismo , Temperatura , Glucose/metabolismoRESUMO
Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 â 3) and ß(1 â 6) elongating activity, but also some ß(1 â 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: ⢠ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. ⢠Thermotoga maritima ß-glucosidase has high activity and high thermostability. ⢠Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.
Assuntos
Celobiose , Lactose , Oligossacarídeos , Thermotoga maritima , beta-Glucosidase , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , Lactose/metabolismo , Celobiose/metabolismo , beta-Glucosidase/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/química , Cinética , Oligossacarídeos/metabolismo , Glicosilação , Hidrólise , Temperatura , Estabilidade EnzimáticaRESUMO
ß-Glucosidase (EC 3.2.1.21) from sweet almond was encapsulated into pH-responsive alginate-polyethylenimine (alginate-PEI) hydrogel. Then, electrochemically controlled cyclic local pH changes resulting from ascorbate oxidation (acidification) and oxygen reduction (basification) were used for the pulsatile release of the enzyme from the composite hydrogel. Activation of the enzyme was controlled by the very same pH changes used for ß-glucosidase release, separating these two processes in time. Importantly, the activity of the enzyme, which had not been released yet, was inhibited due to the buffering effect of PEI present in the gel. Thus, only a portion of the released enzyme was activated. Both enzymatic activity and release were monitored by confocal fluorescence microscopy and regular fluorescent spectroscopy. Namely, commercially available very little or nonfluorescent substrate 4-methylumbelliferyl-ß-d-glucopyranoside was hydrolyzed by ß-glucosidase to produce a highly fluorescent product 4-methylumbelliferone during the activation phase. At the same time, labeling of the enzyme with rhodamine B isothiocyanate was used for release observation. The proposed work represents an interesting smart release-activation system with potential applications in biomedical field.
Assuntos
Alginatos , Hidrogéis , Polietilenoimina , beta-Glucosidase , Alginatos/química , Hidrogéis/química , Polietilenoimina/química , Concentração de Íons de Hidrogênio , beta-Glucosidase/metabolismo , beta-Glucosidase/química , Rodaminas/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Himecromona/química , Ativação Enzimática/efeitos dos fármacos , Prunus/enzimologia , Prunus/química , Ácido Glucurônico/química , Técnicas EletroquímicasRESUMO
A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.
Assuntos
Enzimas Imobilizadas , Sucos de Frutas e Vegetais , Glioxilatos , Sefarose , Sucos de Frutas e Vegetais/análise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Aminação , Concentração de Íons de Hidrogênio , Sefarose/química , Glioxilatos/química , Citrus/química , Citrus/enzimologia , Estabilidade Enzimática , Biocatálise , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Temperatura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flavanonas/química , Flavanonas/metabolismo , CatáliseRESUMO
Limeum indicum has been widely utilized in traditional medicine but no experimental work has been done on this herb. The primary objective of this study was to conduct a phytochemical analysis and assess the multifunctional capabilities of aforementioned plant in dual therapy for Alzheimer's disease (AD) and Typeâ 2 diabetes (T2D). The phytochemical screening of ethanol, methanol extract, and their derived fractions of Limeum indicum was conducted using GC-MS, HPLC, UV-analysis and FTIR. The antioxidant capacity was evaluated by DPPH method. The inhibitory potential of the extracts/fractions against α-, ß-glucosidase acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and monoaminine oxidases (MAO-A & B) was evaluated. Results revealed that acetonitrile fraction has highest inhibitory potential against α-glucosidase (IC50=68.47±0.05â µg/mL), methanol extract against ß-glucosidase (IC50=91.12±0.07â µg/mL), ethyl acetate fraction against AChE (IC50=59.0±0.02â µg/mL), ethanol extract against BChE (28.41±0.01â µg/mL), n-hexane fraction against MAO-A (IC50=150.5±0.31â µg/mL) and methanol extract for MAO-B (IC50=75.95±0.13â µg/mL). The docking analysis of extracts\fractions suggested the best binding scores within the active pocket of the respective enzymes. During the in-vivo investigation, ethanol extract produced hypoglycemic effect (134.52±2.79 and 119.38±1.40â mg/dl) after 21â days treatment at dose level of 250 and 500â mg/Kg. Histopathological findings further supported the in-vivo studies.
Assuntos
Acetilcolinesterase , Doença de Alzheimer , Butirilcolinesterase , Cromatografia Gasosa-Espectrometria de Massas , Hipoglicemiantes , Simulação de Acoplamento Molecular , Monoaminoxidase , Compostos Fitoquímicos , Extratos Vegetais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Animais , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/isolamento & purificação , Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipoglicemiantes/isolamento & purificação , Monoaminoxidase/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/isolamento & purificação , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Masculino , alfa-Glucosidases/metabolismo , Ratos , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/metabolismo , HumanosRESUMO
Flavonoids are usually present in forms of glucosides in plants, which could be catabolized by ß-glucosidase (BGLU) to form their corresponding flavonoid aglycones. In this study, we isolated three abiotic-responsive BGLU genes (MtBGLU17, MtBGLU21 and MtBGLU22) from Medicago truncatula, and found only the recombinant MtBGLU17 protein could catalyse the hydrolysis of flavonoid glycosides. The recombinant MtBGLU17 protein is active towards a variety of flavonoid glucosides, including glucosides of flavones (apigenin and luteolin), flavonols (kaempferol and quercetin), isoflavones (genistein and daidzein) and flavanone (naringenin). In particular, the recombinant MtBGLU17 protein preferentially hydrolyses flavonoid-7-O-glucosides over their corresponding 3-O-glucosides. The content of luteoin-7-O-glucoside was reduced in the MtBGLU17 overexpression plants but increased in the Tnt-1 insertional mutant lines, whereas luteoin content was increased in the MtBGLU17 overexpression plants but reduced in the Tnt-1 insertional mutant lines. Under drought and salt (NaCl) treatment, the MtBGLU17 overexpression lines showed relatively higher DPPH content, and higher CAT and SOD activity than the wild type control. These results indicated that overexpression lines of MtBGLU17 possess higher antioxidant activity and thus confer drought and salt tolerance, implying MtBGLU17 could be potentially used as a candidate gene to improve plant abiotic stress tolerance.
Assuntos
Antioxidantes , Secas , Flavonoides , Medicago truncatula , Proteínas de Plantas , Tolerância ao Sal , beta-Glucosidase , Medicago truncatula/genética , Medicago truncatula/enzimologia , Medicago truncatula/metabolismo , Medicago truncatula/fisiologia , Flavonoides/metabolismo , Antioxidantes/metabolismo , beta-Glucosidase/metabolismo , beta-Glucosidase/genética , Tolerância ao Sal/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de PlantasRESUMO
Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of ß-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in ß-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.
Assuntos
Estabilidade Enzimática , Engenharia de Proteínas , Pyrococcus furiosus , beta-Glucosidase , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , beta-Glucosidase/genética , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Engenharia de Proteínas/métodos , Temperatura BaixaRESUMO
Mesenchymal stem cell (MSC) therapy shows promise in regenerative medicine. For osteoarthritis (OA), MSCs delivered to the joint have a temporal window in which they can secrete growth factors and extracellular matrix molecules, contributing to cartilage regeneration and cell proliferation. However, upon injection in the non-vascularized joint, MSCs lacking energy supply, starve and die too quickly to efficiently deliver enough of these factors. To feed injected MSCs, we developed a hyaluronic acid (HA) derivative, where glucose is covalently bound to hyaluronic acid. To achieve this, the glucose moiety in 4-aminophenyl-ß-D-glucopyranoside was linked to the HA backbone through amidation. The hydrogel was able to deliver glucose in a controlled manner using a trigger system based on hydrolysis catalyzed by endogenous ß-glucosidase. This led to glucose release from the hyaluronic acid backbone inside the cell. Indeed, our hydrogel proved to rescue starvation and cell mortality in a glucose-free medium. Our approach of adding a nutrient to the polymer backbone in hydrogels opens new avenues to deliver stem cells in poorly vascularized, nutrient-deficient environments, such as osteoarthritic joints, and for other regenerative therapies.
Assuntos
Glucose , Ácido Hialurônico , Hidrogéis , Células-Tronco Mesenquimais , Osteoartrite , Ácido Hialurônico/química , Glucose/metabolismo , Osteoartrite/terapia , Hidrogéis/química , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , beta-Glucosidase/metabolismo , AnimaisRESUMO
Environmental problems are caused by the disposal of agrowastes in developing countries. It is imperative to convert such wastes into useful products, which require enzymes such as ß-glucosidase. ß-Glucosidase has variety of applications in biotechnology including food, textile, detergents, pulp and paper, pharmaceutical and biofuel industries. ß-Glucosidase production was performed using the locally isolated Aspergillus protuberus using best growth circumstances on rice husk in solid-state fermentation (SSF). Leaching of ß-glucosidase from fermented rice husk with number of solvents to evaluate their extraction efficacy. Among the different solvents examined, acetate buffer (0.02 M, pH 5.0) proved to be the best solvent. The subsequent parameters were optimized with acetate buffer. Two washes with acetate buffer each by shaking (30 min) in a ratio of 1 g of rice husk: 5 ml of acetate buffer together attained maximum recovery of ß-glucosidase with 41.95 U/g of rice husk.