Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides
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 oft...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/8/4106 |
| _version_ | 1797446007871504384 |
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| author | Pavlína Nekvasilová Michaela Hovorková Zuzana Mészáros Lucie Petrásková Helena Pelantová Vladimír Křen Kristýna Slámová Pavla Bojarová |
| author_facet | Pavlína Nekvasilová Michaela Hovorková Zuzana Mészáros Lucie Petrásková Helena Pelantová Vladimír Křen Kristýna Slámová Pavla Bojarová |
| author_sort | Pavlína Nekvasilová |
| collection | DOAJ |
| description | 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-<i>N</i>-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-<i>N</i>-acetylhexosaminidase BbhI from <i>Bifidobacterium bifidum</i>, β4-galactosidase BgaD-B from <i>Bacillus circulans</i>, β4-<i>N</i>-acetylgalactosaminidase from <i>Talaromyces flavus</i>, and β3-galactosynthase BgaC from <i>B. circulans</i>. 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. |
| first_indexed | 2024-03-09T13:34:13Z |
| format | Article |
| id | doaj.art-5f202e60149f4a7985245b45ceecaca3 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T13:34:13Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-5f202e60149f4a7985245b45ceecaca32023-11-30T21:14:06ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-04-01238410610.3390/ijms23084106Engineered Glycosidases for the Synthesis of Analogs of Human Milk OligosaccharidesPavlína Nekvasilová0Michaela Hovorková1Zuzana Mészáros2Lucie Petrásková3Helena Pelantová4Vladimír Křen5Kristýna Slámová6Pavla Bojarová7Laboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicLaboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicLaboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicLaboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicLaboratory of Molecular Structure Characterization, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicLaboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicLaboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicLaboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha, Czech RepublicEnzymatic 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-<i>N</i>-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-<i>N</i>-acetylhexosaminidase BbhI from <i>Bifidobacterium bifidum</i>, β4-galactosidase BgaD-B from <i>Bacillus circulans</i>, β4-<i>N</i>-acetylgalactosaminidase from <i>Talaromyces flavus</i>, and β3-galactosynthase BgaC from <i>B. circulans</i>. 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.https://www.mdpi.com/1422-0067/23/8/4106enzymatic synthesisglycosidasehuman milk oligosaccharidemutagenesis |
| spellingShingle | Pavlína Nekvasilová Michaela Hovorková Zuzana Mészáros Lucie Petrásková Helena Pelantová Vladimír Křen Kristýna Slámová Pavla Bojarová Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides International Journal of Molecular Sciences enzymatic synthesis glycosidase human milk oligosaccharide mutagenesis |
| title | Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides |
| title_full | Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides |
| title_fullStr | Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides |
| title_full_unstemmed | Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides |
| title_short | Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides |
| title_sort | engineered glycosidases for the synthesis of analogs of human milk oligosaccharides |
| topic | enzymatic synthesis glycosidase human milk oligosaccharide mutagenesis |
| url | https://www.mdpi.com/1422-0067/23/8/4106 |
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