Glycosidase Dotaz Zobrazit nápovědu
Natural flavonoids, especially in their glycosylated forms, are the most abundant phenolic compounds found in plants, fruit, and vegetables. They exhibit a large variety of beneficial physiological effects, which makes them generally interesting in a broad spectrum of scientific areas. In this review, we focus on recent advances in the modifications of the glycosidic parts of various flavonoids employing glycosidases, covering both selective trimming of the sugar moieties and glycosylation of flavonoid aglycones by natural and mutant glycosidases. Glycosylation of flavonoids strongly enhances their water solubility and thus increases their bioavailability. Antioxidant and most biological activities are usually less pronounced in glycosides, but some specific bioactivities are enhanced. The presence of l-rhamnose (6-deoxy-α-l-mannopyranose) in rhamnosides, rutinosides (rutin, hesperidin) and neohesperidosides (naringin) plays an important role in properties of flavonoid glycosides, which can be considered as "pro-drugs". The natural hydrolytic activity of glycosidases is widely employed in biotechnological deglycosylation processes producing respective aglycones or partially deglycosylated flavonoids. Moreover, deglycosylation is quite commonly used in the food industry aiming at the improvement of sensoric properties of beverages such as debittering of citrus juices or enhancement of wine aromas. Therefore, natural and mutant glycosidases are excellent tools for modifications of flavonoid glycosides.
- MeSH
- flavonoidy metabolismus MeSH
- glykosidhydrolasy metabolismus MeSH
- isoflavony metabolismus MeSH
- katechin metabolismus MeSH
- lidé MeSH
- quercetin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
A novel endophytic fungus producing beta-glucosidase was isolated and characterized from pigeon pea (Cajanus cajan [L.] Millsp.), which has excellent properties in converting ginsenoside Rb1 to ginsenoside Rd in Panax notoginseng. According to the 16S rDNA gene sequence, the G11-7 strain was identified as Fusarium proliferatum, and the accession number KY303906 was confirmed in GenBank. The G11-7 immobilized spores, in which the activity of beta-glucosidase could reach 0.95 U/mL, were co-cultured with P. notoginseng plant material to obtain a continuous beta-glucosidase supply for the biotransformation of ginsenoside Rb1 to Rd. Under the liquid-solid ratio (20:1), initial pH (6.0), and temperature (30 °C) constituents, the maximum ginsenoside Rd yield was obtained as 9.15 ± 0.65 mg/g, which was 3.67-fold higher than that without fungal spore co-culture (2.49 ± 0.98 mg/g). Furthermore, immobilized G11-7 spores showed significant beta-glucosidase producing ability which could be recovered and reused for 6 cycles. Overall, these results suggested that immobilized G11-7 offered a promising and effective approach to enhance the production of ginsenoside Rd for possible nutraceutical and pharmaceutical uses.
In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors.
- MeSH
- elektroforéza kapilární metody MeSH
- enzymy imobilizované chemie metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- glykosylace MeSH
- imunoglobulin G chemie MeSH
- lidé MeSH
- mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa chemie metabolismus MeSH
- polysacharidy analýza chemie MeSH
- poréznost MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A novel class of naturally-occurring glycosidase inhibitors, having sulfonium sulfate structures, has been isolated as bioactive components from Indian plants, belonging to the Salacia genus in the family Celastraceae, and used in Ayurvedic medicine for the treatment of type-2 diabetes. Thus far, five such sulfonium salts, namely, salacinol, kotalanol, salaprinol, ponkoranol and de-O-sulfonated kotalanol, have been isolated from this plant species. These structurally unique zwitterionic glycosidase inhibitors have received much attention due to their therapeutic potential in the treatment of type-2 diabetes. We recently reported a review article which focused mainly on salacinol and related analogues. The present review presents an update on the remaining four compounds from this class of glycosidase inhibitors, with respect to their isolation, glucosidase inhibitory activities, and synthesis. In addition, progress towards the stereochemical structure elucidation of kotalanol, through synthesis of analogues, is described. Review with 42 references.
Thanks to the stability, good availability, stereoselectivity and broad substrate specificity, oligosaccharide synthesis catalyzed by glycosidases represents an elegant way to complex carbohydrate structures. Two approaches to glycosidase catalysis are presented: (i) the use of structurally modified substrates that carry various functional moieties in the molecule, and (ii) the design of mutant glycosidases void of hydrolytic activity. Products of glycosidase-catalyzed synthesis are applicable in a range of areas such as immunology, therapy of Alzheimer's or Parkinson's diseases and the synthesis of neoglycoproteins.
In recent years, carbohydrate-processing enzymes have become the enzymes of choice in many applications thanks to their stereoselectivity and efficiency. This review presents recent developments in glycosidase-catalyzed synthesis via two complementary approaches: the use of wild-type enzymes with engineered substrates, and mutant glycosidases. Genetic engineering has recently produced glucuronyl synthases, an inverting xylosynthase and the first mutant endo-beta-N-acetylglucosaminidase. A thorough selection of enzyme strains and aptly modified substrates have resulted in rare glycostructures, such as N-acetyl-beta-galactosaminuronates, beta1,4-linked mannosides and alpha1,4-linked galactosides. The efficient selection of mutant enzymes is facilitated by high-throughput screening assays involving the co-expression of coupled enzymes or chemical complementation. Selective glycosidase inhibitors and highly specific glycosidases are finding attractive applications in biomedicine, biology and proteomics.
BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.
- MeSH
- beta-N-acetylhexosaminidasy chemie metabolismus MeSH
- fylogeneze MeSH
- glykosylace MeSH
- katalytická doména MeSH
- kinetika MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- reprodukovatelnost výsledků MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- substrátová specifita MeSH
- Talaromyces enzymologie MeSH
- výpočetní biologie * MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Until recently, glycosidases, naturally hydrolyzing carbohydrate-active enzymes, have found few synthetic applications in industry, being primarily used for cleaving unwanted carbohydrates. With the establishment of glycosynthase and transglycosidase technology by genetic engineering, the view of glycosidases as industrial biotechnology tools has started to change. Their easy production, affordability, robustness, and substrate versatility, added to the possibility of controlling undesired side hydrolysis by enzyme engineering, have made glycosidases competitive synthetic tools. Current promising applications of engineered glycosidases include the production of well-defined chitooligomers, precious galactooligosaccharides or specialty chemicals such as glycosylated flavonoids. Other synthetic pathways leading to human milk oligosaccharides or remodeled antibodies are on the horizon. This work provides an overview of the synthetic achievements to date for glycosidases, emphasizing the latest trends and outlining possible developments in the field.
- MeSH
- glykosidhydrolasy * genetika metabolismus MeSH
- glykosylace MeSH
- hydrolýza MeSH
- lidé MeSH
- oligosacharidy * MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Until recently, glycosidases, naturally hydrolyzing carbohydrate-active enzymes, have found few synthetic applications in industry, being primarily used for cleaving unwanted carbohydrates. With the establishment of glycosynthase and transglycosidase technology by genetic engineering, the view of glycosidases as industrial biotechnology tools has started to change. Their easy production, affordability, robustness, and substrate versatility, added to the possibility of controlling undesired side hydrolysis by enzyme engineering, have made glycosidases competitive synthetic tools. Current promising applications of engineered glycosidases include the production of well-defined chitooligomers, precious galactooligosaccharides or specialty chemicals such as glycosylated flavonoids. Other synthetic pathways leading to human milk oligosaccharides or remodeled antibodies are on the horizon. This work provides an overview of the synthetic achievements to date for glycosidases, emphasizing the latest trends and outlining possible developments in the field.
Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.
- MeSH
- Bifidobacterium bifidum * metabolismus MeSH
- glykosidhydrolasy metabolismus MeSH
- glykosyltransferasy metabolismus MeSH
- lidé MeSH
- mateřské mléko * metabolismus MeSH
- novorozenec MeSH
- oligosacharidy chemie MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
