Characterization and immobilization of engineered sialidases from <em>Trypanosoma rangeli</em> for transsialylation

Birgitte Zeuner; Isabel González-Delgado; Jesper Holck; Gabriel Morales; María-José López-Muñoz; Yolanda Segura; Anne S. Meyer; Jørn Dalgaard Mikkelsen; Birgitte Zeuner; Isabel González-Delgado; Jesper Holck; Gabriel Morales; María-José López-Muñoz; Yolanda Segura; Anne S. Meyer; Jørn Dalgaard Mikkelsen

doi:10.3934/molsci.2017.2.140

AIMS Molecular Science

2017, Volume 4, Issue 2: 140-163. doi: 10.3934/molsci.2017.2.140

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Characterization and immobilization of engineered sialidases from Trypanosoma rangeli for transsialylation

1.
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
2.
Chemical and Environmental Engineering Group, ESCET, Universidad Rey Juan Carlos, c/Tulipán s/n, 28933 Móstoles, Madrid, Spain

Received: 13 January 2017 Accepted: 06 April 2017 Published: 13 April 2017

A sialidase (EC 3.2.1.18; GH 33) from non-pathogenic Trypanosoma rangeli 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_5mut. 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 T. rangeli sialidase variants and higher than that of the native transsialidase from T. cruzi (TcTS). As for other engineered T. rangeli sialidase variants and TcTS, the acceptor specificity was broad: lactose, galactooligosaccharides (GOS), xylooligosaccharides (XOS), and human milk oligosaccharide structures lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP V), and lacto-N-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.
- Transsialylation,
- transsialidase,
- Trypanosoma rangeli,
- enzyme immobilization,
- casein glycomacropeptide (CGMP),
- GH33,
- human milk oligosaccharides (HMOs),
- galactooligosaccharides (GOS)
Citation: 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 Trypanosoma rangeli for transsialylation[J]. AIMS Molecular Science, 2017, 4(2): 140-163. doi: 10.3934/molsci.2017.2.140

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Abstract

A sialidase (EC 3.2.1.18; GH 33) from non-pathogenic Trypanosoma rangeli 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_5mut. 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 T. rangeli sialidase variants and higher than that of the native transsialidase from T. cruzi (TcTS). As for other engineered T. rangeli sialidase variants and TcTS, the acceptor specificity was broad: lactose, galactooligosaccharides (GOS), xylooligosaccharides (XOS), and human milk oligosaccharide structures lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP V), and lacto-N-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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