β-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective β-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal β-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant β-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 - a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications.
- MeSH
- Aspergillus enzymologie MeSH
- beta-N-acetylhexosaminidasy chemie genetika izolace a purifikace metabolismus MeSH
- chromatografie iontoměničová MeSH
- disacharidy metabolismus MeSH
- exprese genu MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- Pichia genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- Talaromyces enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
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
BACKGROUND: In recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides. SCOPE OF REVIEW: This review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, β-N-acetylhexosaminidases, and endo-β-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans. MAJOR CONCLUSIONS: The above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions. GENERAL SIGNIFICANCE: Specific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good understanding of its details opens new possibilities in this fast developing area of glycoscience.
- MeSH
- glykoproteiny biosyntéza MeSH
- glykosidhydrolasy metabolismus MeSH
- glykosylace MeSH
- glykosyltransferasy metabolismus MeSH
- hexosaminy metabolismus MeSH
- katalýza MeSH
- oligosacharidy biosyntéza MeSH
- proteinové inženýrství * MeSH
- sulfotransferasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
