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

doi:10.3934/molsci.2017.2.140

AIMS Molecular Science, 2017, 4(2): 140-163. doi: 10.3934/molsci.2017.2.140. Research article Topical Section

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Characterization and immobilization of engineered sialidases from Trypanosoma rangeli for transsialylation

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

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

† These two authors contributed equally.

Received: , Accepted: , Published:

Topical Section: Enzyme Activity and Immobilization

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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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Keywords: 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. AIMS Molecular Science, 2017, 4(2): 140-163. doi: 10.3934/molsci.2017.2.140

References:

1. Bode L (2012) Human milk oligosaccharides: Every baby needs a sugar mama. Glycobiology 22: 1147-1162.
2. Kunz C, Meyer C, Collado MC, et al. (2016) Influence of gestational age, secretor and Lewis blood group status on the oligosaccharide content of human milk. J Pediatr Gastroenterol Nutr in press.
3. ten Bruggencate SJM, Bovee-Oudenhoven IMJ, Feitsma AL, et al. (2014) Functional role and mechanisms of sialyllactose and other sialylated milk oligosaccharides. Nutr Rev 72: 377-389.
4. Holck J, Larsen DM, Michalak M, et al. (2014) Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase. New Biotechnol 31: 156-165.
5. Wilbrink MH, ten Kate GA, van Leeuwen SS, et al. (2014) Galactosyl-lactose sialylation using Trypanosoma cruzi trans-sialidase as the biocatalyst and bovine κ-casein-derived glycomacropeptide as the donor substrate. Appl Environ Microbiol 80: 5984-5991.
6. Wilbrink MH, ten Kate GA, Sanders P, et al. (2015) Enzymatic decoration of prebiotic galacto-oligosaccharides (Vivinal GOS) with sialic acid using Trypanosoma cruzi trans-sialidase and two bovine sialoglycoconjugates as donor substrates. J Agric Food Chem 63: 5976-5984.
7. Scudder P, Doom JP, Chuenkova M, et al. (1993) Enzymatic characterization of β-D-galactoside α2,3-transsialidase from Trypanosoma cruzi. J Biol Chem 268: 9886-9891.
8. Pereira ME, Zhang K, Gong Y, et al. (1996) Invasive phenotype of Trypanosoma cruzi restricted to a population expressing trans-sialidase. Infect Immun 64: 3884-3892.
9. Paris G, Ratier L, Amaya MF, et al. (2005) A sialidase mutant displaying trans-sialidase activity. J Mol Biol 345: 923-934.
10. Jers C, Michalak M, Larsen DM, et al. (2014) Rational design of a new Trypanosoma rangeli trans-sialidase for efficient sialylation of glycans. PLoS One 9: e83902.
11. Pontes-de-Carvalho LC, Tomlinson S, Nussenzweig V (1993) Trypanosoma rangeli sialidase lacks trans-sialidase activity. Mol Biochem Parasitol 62: 19-25.
12. Amaya MF, Buschiazzo A, Nguyen T, et al. (2003) The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase. J Mol Biol 325: 773-784.
13. Buschiazzo A, Tavares GA, Campetella O, et al. (2000) Structural basis of sialyltransferase activity in trypanosomal sialidases. EMBO J 19: 16-24.
14. Pierdominici-Sottile G, Palma J, Roitberg AE (2014) Free-energy computations identify the mutations required to confer trans-sialidase activity into Trypanosoma rangeli sialidase. Proteins 82: 424-435.
15. Zeuner B, Luo J, Nyffenegger C, et al. (2014) Optimizing the biocatalytic productivity of an engineered sialidase from Trypanosoma rangeli for 3'-sialyllactose production. Enzyme Microb Technol 55: 85-93.
16. Michalak M, Larsen DM, Jers C, et al. (2014) Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity. Process Biochem 49: 265-270.
17. Zeuner B, Holck J, Perna V, et al. (2016) Quantitative enzymatic production of sialylated galactooligosaccharides with an engineered sialidase from Trypanosoma rangeli. Enzyme Microb Technol 82: 42-50.
18. Nyffenegger C, Nordvang RT, Jers C, et al. (2017) Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity. PLoS One 12: e0171585.
19. Kasche V (1986) Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of β-lactam antibiotics, peptides and other condensation products. Enzyme Microb Technol 8: 4-16.
20. van Rantwijk F, Woudenberg-van Oosterom M, Sheldon RA (1999) Glycosidase-catalysed synthesis of alkyl glycosides. J Mol Catal B-Enzym 6: 511-532.
21. Hansson T, Andersson M, Wehtje E, et al. (2001) Influence of water activity on the competition between β-glycosidase catalysed transglycosylation and hydrolysis in aqueous hexanol. Enzyme Microb Technol 29: 527-534.
22. Zeuner B, Jers C, Mikkelsen JD, et al. (2014) Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics. J Agric Food Chem 62: 9615-9631.
23. Mateo C, Palomo JM, Fernandez-Lorente G, et al. (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 40: 1451-1463.
24. Rodrigues RC, Ortiz C., Berenguer-Murcia A, et al. (2013) Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev 42: 6290-6307.
25. Barbosa O, Ortiz C, Berenguer-Murcia A, et al. (2014) Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv 4: 1583-1600.
26. Grimsley GR, Scholtz JM, Pace CN (2009) A summary of the measured pK values of the ionizable groups in folded proteins. Protein Sci 18: 247-251.
27. Mateo C, Abian O, Bernedo M, et al. (2005) Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb Technol 37: 456-462.
28. Mateo C, Palomo JM, Fuentes M, et al. (2006) Glyoxyl agarose: A fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme Microb Technol 39: 274-280.
29. Barbosa O, Ortiz C, Berenguer-Murcia A, et al. (2015) Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts. Biotechnol Adv 33: 435-456.
30. Zucca P, Fernandez-Lafuente R, Sanjust E (2016) Agarose and its derivatives as supports for enzyme immobilization. Molecules 21: 1577.
31. Calandri C, Marques DP, Mateo C, et al. (2013) Purification, immobilization, stabilization and characterization of commercial extract with β-galactosidase activity. J Biocatal Biotransformation 2: 1-7.
32. Hartmann M, Kostrov X (2013) Immobilization of enzymes on porous silicas – benefits and challenges. Chem Soc Rev 42: 6277-6289.
33. Bernal C, Urrutia P, Illanes A, et al. (2013) Hierarchical meso-macroporous silica grafted with glyoxyl groups: opportunities for covalent immobilization of enzymes. New Biotechnol 30: 500-506.
34. Bernal C, Sierra L, Mesa M (2014) Design of β-galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance. Eng Life Sci 14: 85-94.
35. González-Delgado I, Segura Y, Morales G, et al. (2017) Production of high galacto-oligosaccharides by Pectinex Ultra SP-L: optimization of reaction conditions and immobilization on glyoxyl-functionalized silica. J Agric Food Chem 65: 1649-1658.
36. Liu Y, Li Y, Li XM, et al. (2013) Kinetics of (3-aminopropyl)triethoxysilane (APTES) silanization of superparamagnetic iron oxide nanoparticles. Langmuir 29: 15275-15282.
37. Gunda NSK, Singh M, Norman L, et al. (2014) Optimization and characterization of biomolecule immobilization on silicon substrates using (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde linker. Appl Surf Sci 305: 522-530.
38. Zhang D, Hegab HE, Lvov Y, et al. (2016) Immobilization of cellulase on a silica gel substrate modified using a 3-APTES self-assembled monolayer. SpringerPlus 5: 48.
39. Nordvang RT, Nyffenegger C, Holck J, et al. (2016) It all starts with a sandwich: Identification of sialidases with trans-glycosylation activity. PLoS One 11: e0158434.
40. Alva V, Nam SZ, Söding J, et al. (2016) The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis. Nucleic Acids Res 44: W410-W415.
41. Sayle R, Milner-White EJ (1995) RasMol: Biomolecular graphics for all. Trends Biochem Sci 20: 374-376.
42. Fersht AR, Serrano L (1993) Principles of protein stability derived from protein engineering experiments. Curr Opin Struct Biol 3: 75-83.
43. Torrez M, Schultehenrich M, Livesay DR (2003) Conferring thermostability to mesophilic proteins through optimized electrostatic surfaces. Biophys J 85: 2845-2853.
44. Hagiwara Y, Sieverling L, Hanif F, et al. (2016) Consequences of point mutations in melanoma-associated antigen 4 (MAGE-A4) protein: Insights from structural and biophysical studies. Sci Rep 6: 25182.
45. Lu Y, Zen KC, Muthukrishnan S, et al. (2002) Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142, D144 and E146 in Manduca sexta (tobacco hornworm) chitinase. Insect Biochem Mol Biol 32: 1369-1382.
46. Cha J, Batt CA (1998) Lowering the pH optimum of D-xylose isomerase: the effect of mutations of the negatively charged residues. Mol Cells 8: 374-382.
47. Joshi MD, Sidhu G, Pot I, et al. (2000) Hydrogen bonding and catalysis: A novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J Mol Biol 299: 255-279.
48. Hirata A, Adachi M, Sekine A, et al. (2004) Structural and enzymatic analysis of soybean β-amylase mutants with increased pH optimum. J Biol Chem 279: 7287-7295.
49. Amaya MF, Watts AG, Damager I, et al. (2004) Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Structure 12: 775-784.
50. Vandekerckhove F, Schenkman S, Pontes de Carvalho L, et al. (1992) Substrate specificity of the Trypanosoma cruzi trans-sialidase. Glycobiology 2: 541-548.
51. Bridiau N, Issaoui N, Maugard T (2010) The effects of organic solvents on the efficiency and regioselectivity of N-acetyl-lactosamine synthesis, using the β-galactosidase from Bacillus circulans in hydro-organic media. Biotechnol Prog 26: 1278-1289.
52. Thiem J, Sauerbrei B (1991) Chemoenzymatic syntheses of sialyloligosaccharides with immobilized sialidase. Angew Chem Int Ed Engl 30: 1503-1505.
53. Ajisaka K, Fujimoto H, Isomura M (1994) Regioselective transglycosylation in the synthesis of oligosaccharides: comparison of β-galactosidases and sialidases of various origins. Carbohydr Res 259: 103-115.
54. Marques ME, Mansur AAP, Mansur HS (2013) Chemical functionalization of surfaces for building three-dimensional engineered biosensors. Appl Surf Sci 275: 347-360.
55. Ferreira L, Ramos MA, Dordick JS, et al. (2003) Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg). J Mol Catal B-Enzym 21: 189-199.
56. Thomä-Worringer C, Sørensen J, López-Fandiño R (2006) Health effect and technological features of caseinomacropeptide. Int Dairy J 16:1324-1333.
57. Koshland D (1953) Stereochemistry and the mechanism of enzymatic reactions. Biol Rev 28: 416-436.

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This article has been cited by:

1. Birgitte Zeuner, David Teze, Jan Muschiol, Anne S. Meyer, Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation, Molecules, 2019, 24, 11, 2033, 10.3390/molecules24112033

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