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
- Glycoside Hydrolases * genetics metabolism MeSH
- Glycosylation MeSH
- Hydrolysis MeSH
- Humans MeSH
- Oligosaccharides * MeSH
- Substrate Specificity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
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.
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.
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.
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 * metabolism MeSH
- Glycoside Hydrolases metabolism MeSH
- Glycosyltransferases metabolism MeSH
- Humans MeSH
- Milk, Human * metabolism MeSH
- Infant, Newborn MeSH
- Oligosaccharides chemistry MeSH
- Substrate Specificity MeSH
- Check Tag
- Humans MeSH
- Infant, Newborn MeSH
- Publication type
- Journal Article MeSH
Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, β-d-galactosidase, β-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and β-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and β-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both β-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.
- MeSH
- Biotinylation MeSH
- Protein Array Analysis MeSH
- Follicular Fluid chemistry MeSH
- Glycoside Hydrolases blood chemistry metabolism MeSH
- Oocytes MeSH
- Swine * MeSH
- Seminal Plasma Proteins chemistry metabolism MeSH
- Zona Pellucida chemistry physiology MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- MeSH
- alpha-Glucosidases diagnostic use MeSH
- beta-Galactosidase diagnostic use MeSH
- Chromatography, Affinity * methods utilization MeSH
- Electrophoresis, Polyacrylamide Gel methods utilization MeSH
- Glucan 1,4-alpha-Glucosidase diagnostic use MeSH
- Glycoside Hydrolases * isolation & purification metabolism MeSH
- Rats MeSH
- Lactase diagnostic use MeSH
- Sepharose diagnostic use isolation & purification MeSH
- Statistics as Topic MeSH
- Intestinal Mucosa enzymology MeSH
- Intestine, Small * enzymology metabolism MeSH
- Thioglucosides diagnostic use MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- MeSH
- Aorta enzymology metabolism MeSH
- Arteries enzymology metabolism pathology MeSH
- Plaque, Atherosclerotic diagnosis enzymology etiology MeSH
- beta-Galactosidase diagnostic use metabolism MeSH
- Endothelium, Vascular * enzymology metabolism pathology MeSH
- Glucosidases isolation & purification metabolism MeSH
- Glucuronidase diagnostic use metabolism MeSH
- Glycoside Hydrolases * isolation & purification metabolism MeSH
- Histocytochemistry methods utilization MeSH
- Clinical Enzyme Tests methods utilization MeSH
- Coronary Vessels * enzymology metabolism MeSH
- Rabbits MeSH
- Chickens MeSH
- Humans MeSH
- Guinea Pigs MeSH
- Rats, Wistar MeSH
- Statistics as Topic MeSH
- Check Tag
- Rabbits MeSH
- Humans MeSH
- Guinea Pigs MeSH
