Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation
A sialidase (EC 3.2.1.18; GH 33) from non-pathogenic <em>Trypanosoma rangeli</em> has been engineered with the aim of improving its transsialylation activity. Recently, two engineered variants containing 15 and 16 amino acid substitutions, respectively, were found to exhibit significantl...
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| Format: | Article |
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AIMS Press
2017-04-01
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| Series: | AIMS Molecular Science |
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| Online Access: | http://www.aimspress.com/Molecular/article/1376/fulltext.html |
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| author | Birgitte Zeuner Isabel González-Delgado Jesper Holck Gabriel Morales María-José López-Muñoz Yolanda Segura Anne S. Meyer Jørn Dalgaard Mikkelsen |
| author_facet | Birgitte Zeuner Isabel González-Delgado Jesper Holck Gabriel Morales María-José López-Muñoz Yolanda Segura Anne S. Meyer Jørn Dalgaard Mikkelsen |
| author_sort | Birgitte Zeuner |
| collection | DOAJ |
| description | A sialidase (EC 3.2.1.18; GH 33) from non-pathogenic <em>Trypanosoma rangeli</em> has been engineered with the aim of improving its transsialylation activity. Recently, two engineered variants containing 15 and 16 amino acid substitutions, respectively, were found to exhibit significantly improved transsialylation activity: both had a 14 times higher ratio between transsialylation and hydrolysis products compared to the first reported mutant TrSA<sub>5mut</sub>. In the current work, these two variants, Tr15 and Tr16, were characterized in terms of pH optimum, thermal stability, effect of acceptor-to-donor ratio, and acceptor specificity for transsialylation using casein glycomacropeptide (CGMP) as sialyl donor and lactose or other human milk oligosaccharide core structures as acceptors. Both sialidase variants exhibited pH optima around pH 4.8. Thermal stability of each enzyme was comparable to that of previously developed <em>T. rangeli </em>sialidase variants and higher than that of the native transsialidase from <em>T. cruzi </em>(TcTS). As for other engineered <em>T. rangeli </em>sialidase variants and TcTS, the acceptor specificity was broad: lactose, galactooligosaccharides (GOS), xylooligosaccharides (XOS), and human milk oligosaccharide structures lacto-<em>N</em>-tetraose (LNT), lacto-<em>N</em>-fucopentaose (LNFP V), and lacto-<em>N</em>-neofucopentaose V (LNnFP V) were all sialylated by Tr15 and Tr16. An increase in acceptor-to-donor ratio from 2 to 10 had a positive effect on transsialylation. Both enzymes showed high preference for formation α(2,3)-linkages at the non-reducing end of lactose in the transsialylation. Tr15 was the most efficient enzyme in terms of transsialylation reaction rates and yield of 3’-sialyllactose. Finally, Tr15 was immobilized covalently on glyoxyl-functionalized silica, leading to a 1.5-fold increase in biocatalytic productivity (mg 3’-sialyllactose per mg enzyme) compared to free enzyme after 6 cycles of reuse. The use of glyoxyl-functionalized silica proved to be markedly better for immobilization than silica functionalized with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde, which resulted in a biocatalytic productivity which was less than half of that obtained with free enzyme. |
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| institution | Directory Open Access Journal |
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| spelling | doaj.art-b974c94ccfba40c8a0f13d1fff183e512022-12-22T03:41:52ZengAIMS PressAIMS Molecular Science2372-03012017-04-014214016310.3934/molsci.2017.2.140molsci-04-00140Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylationBirgitte Zeuner0Isabel González-Delgado1Jesper Holck2Gabriel Morales3María-José López-Muñoz4Yolanda Segura5Anne S. Meyer6Jørn Dalgaard Mikkelsen7Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800 Kgs. Lyngby, DenmarkChemical and Environmental Engineering Group, ESCET, Universidad Rey Juan Carlos, c/Tulipán s/n, 28933 Móstoles, Madrid, SpainCenter for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800 Kgs. Lyngby, DenmarkChemical and Environmental Engineering Group, ESCET, Universidad Rey Juan Carlos, c/Tulipán s/n, 28933 Móstoles, Madrid, SpainChemical and Environmental Engineering Group, ESCET, Universidad Rey Juan Carlos, c/Tulipán s/n, 28933 Móstoles, Madrid, SpainChemical and Environmental Engineering Group, ESCET, Universidad Rey Juan Carlos, c/Tulipán s/n, 28933 Móstoles, Madrid, SpainCenter for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800 Kgs. Lyngby, DenmarkCenter for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800 Kgs. Lyngby, DenmarkA sialidase (EC 3.2.1.18; GH 33) from non-pathogenic <em>Trypanosoma rangeli</em> has been engineered with the aim of improving its transsialylation activity. Recently, two engineered variants containing 15 and 16 amino acid substitutions, respectively, were found to exhibit significantly improved transsialylation activity: both had a 14 times higher ratio between transsialylation and hydrolysis products compared to the first reported mutant TrSA<sub>5mut</sub>. In the current work, these two variants, Tr15 and Tr16, were characterized in terms of pH optimum, thermal stability, effect of acceptor-to-donor ratio, and acceptor specificity for transsialylation using casein glycomacropeptide (CGMP) as sialyl donor and lactose or other human milk oligosaccharide core structures as acceptors. Both sialidase variants exhibited pH optima around pH 4.8. Thermal stability of each enzyme was comparable to that of previously developed <em>T. rangeli </em>sialidase variants and higher than that of the native transsialidase from <em>T. cruzi </em>(TcTS). As for other engineered <em>T. rangeli </em>sialidase variants and TcTS, the acceptor specificity was broad: lactose, galactooligosaccharides (GOS), xylooligosaccharides (XOS), and human milk oligosaccharide structures lacto-<em>N</em>-tetraose (LNT), lacto-<em>N</em>-fucopentaose (LNFP V), and lacto-<em>N</em>-neofucopentaose V (LNnFP V) were all sialylated by Tr15 and Tr16. An increase in acceptor-to-donor ratio from 2 to 10 had a positive effect on transsialylation. Both enzymes showed high preference for formation α(2,3)-linkages at the non-reducing end of lactose in the transsialylation. Tr15 was the most efficient enzyme in terms of transsialylation reaction rates and yield of 3’-sialyllactose. Finally, Tr15 was immobilized covalently on glyoxyl-functionalized silica, leading to a 1.5-fold increase in biocatalytic productivity (mg 3’-sialyllactose per mg enzyme) compared to free enzyme after 6 cycles of reuse. The use of glyoxyl-functionalized silica proved to be markedly better for immobilization than silica functionalized with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde, which resulted in a biocatalytic productivity which was less than half of that obtained with free enzyme.http://www.aimspress.com/Molecular/article/1376/fulltext.htmlTranssialylationtranssialidaseTrypanosoma rangelienzyme immobilizationcasein glycomacropeptide (CGMP)GH33human milk oligosaccharides (HMOs)galactooligosaccharides (GOS) |
| spellingShingle | Birgitte Zeuner Isabel González-Delgado Jesper Holck Gabriel Morales María-José López-Muñoz Yolanda Segura Anne S. Meyer Jørn Dalgaard Mikkelsen Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation AIMS Molecular Science Transsialylation transsialidase Trypanosoma rangeli enzyme immobilization casein glycomacropeptide (CGMP) GH33 human milk oligosaccharides (HMOs) galactooligosaccharides (GOS) |
| title | Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation |
| title_full | Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation |
| title_fullStr | Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation |
| title_full_unstemmed | Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation |
| title_short | Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation |
| title_sort | characterization and immobilization of engineered sialidases from em trypanosoma rangeli em for transsialylation |
| topic | Transsialylation transsialidase Trypanosoma rangeli enzyme immobilization casein glycomacropeptide (CGMP) GH33 human milk oligosaccharides (HMOs) galactooligosaccharides (GOS) |
| url | http://www.aimspress.com/Molecular/article/1376/fulltext.html |
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