Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Transglutaminase-catalysed glycosidation of trypsin with aminated polysaccharides

  • 102 Accesses

  • 9 Citations


Dextran (MW=7.2×104), carboxymethylcellulose (MW=2.5×104, substitution degree=0.7) and Ficoll (MW=6.9×104) were derivatized with 1,4-diaminobutane and covalently attached to bovine pancreatic trypsin through a transglutaminase-catalysed reaction. The conjugates contained an average of 0.7–1.8 mol of polymers per mol of protein, and retained about 61–82% of the original esterolytic activity of trypsin. The optimum pH for trypsin was shifted to alkaline values after enzymatic glycosidation. The thermostability of the polymer–enzyme complexes was increased in about 13.7–50.0 °C over 10 min incubation. The prepared conjugates were also more stable against thermal incubation at different temperatures ranging from 50 °C to 60 °C. In comparison with native trypsin, the enzyme-polymer complexes were about 22- to 48-fold more resistant to autolytic degradation at pH 9.0. Transglutaminase-catalysed glycosidation also protected trypsin against denaturation in surfactant media, with 9- to 68–fold increased half-life times in the presence of 0.3% (w/v) sodium dodecylsulfate.

This is a preview of subscription content, log in to check access.


  1. Aeschlimann D., Paulsson M., 1994 Transglutaminases: protein cross-linking enzymes in tissues and body fluids Thrombosis and Haemostasis 71:402–415

  2. Ando H., Adachi M., Umeda K., Matsuura A., Nonaka M., Uchio K., Tanaka H., Motoki M., 1989 Purification and characteristics of a novel transglutaminase derived from microorganisms Agricultural and Biological Chemistry 53:2613–2617

  3. Blomhoff H.K., Christensen T.B., 1983 Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis β-galactosidase and human serum albumin Biochimica et Biophysica Acta. 743:401–407

  4. Bruneel D., Schacht E., 1993 Chemical modification of pullulan: 1. Periodate oxidation Polymer 34:2628–2632

  5. Chen J.P., Hsu M.S., 1997 Preparation and properties of temperature-sensitive poly(N-isopropylacrylamide)-chymotrypsin conjugates Journal of Molecular Catalysis B Enzymatic 2:233–241

  6. Darias R., Villalonga R., 2001 Functional stabilization of cellulase by covalent modification with chitosan Journal of Chemical Technology and Biotechnology 76:489–493

  7. Darias R., Herrera I., Fragoso A., Cao R., Villalonga R., 2002 Supramolecular interactions mediated thermal stabilization for α-amylase modified with a β-cyclodextrin-carboxymethylcellulose polymer Biotechnology Letters 24:1665–1668

  8. Dubois M.K., Gilles A., Hamilton J.K., Rebers P.A., Smith F., 1956 Colorimetric method for determination of sugars and related substances Analytical Chemistry 28:350–356

  9. Ericsson B., Hegg P.O., 1987 Effects of amphiphiles on trypsin activity and conformation Journal of Dispersion Science and Technology 8:289–301

  10. Esposito C., Costa C., Amoresano A., Mariniello L., Sommella M.G., Caputo I., Porta R., 1999 Transglutaminase-mediated amine incorporation into substance P protects the peptide against proteolysis in vitro Regulatory Peptides 84:75–80

  11. Esposito C., Mancuso F., Calignano A., Di Pierro P., Pucci P., Porta R., 1995 Neurokinin receptors could be differentiated by their capacity to respond to the transglutaminase-synthesized γ-(glutamyl5) spermidine derivative of substance P Journal of Neurochemistry 65:420–426

  12. Folk J.E., Chung S.I., 1985 Transglutaminases Methods in Enzymology 113:358–364

  13. Gómez L., Villalonga R., 2000 Functional stabilization of invertase by covalent modification with pectin Biotechnology Letters 22:1191–1195

  14. Gómez L., Ramírez H.L., Villalonga R., 2000 Stabilization of invertase by modification of sugar chains with chitosan Biotechnology Letters 22:347–350

  15. Gómez L., Ramírez H.L., Villalonga R., 2001 Chemical modification of α-amylase by sodium alginate Acta Biotechnologica 21:265–273

  16. Ho F.F.L., Klosiewics D.W., 1980 Proton nuclear magnetic resonance spectrometry for determination of substituents and their distribution in carboxymethylcellulose Analytical Chemistry 52:913–916

  17. Kanaji T., Ozaki H., Takao T., Kawajiri H., Ide H., Motoki M., Shimonishi Y., 1993 Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112 Journal of Biological Chemistry 268:11565–11572

  18. Klibanov A.M., 1983 Stabilization of enzymes against thermal inactivation Advances in Applied Microbiology 29:1–28

  19. Laemmli U.K., 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227:680–685

  20. Laskowski M., 1955 Trypsinogen and trypsin Methods in Enzymology 2:26–36

  21. Mancuso F., Calignano A., Cozzolino A., Metafora S., Porta R., 1996 Inhibition of zymosan-induced air-pouch inflammation by rat seminal vesicle protein and by its spermidine derivative European Journal of Pharmacology 312:327–332

  22. Mancuso F., Costa C., Calignano A., Mariniello L., Rossi F., Porta R., Esposito C., 1998 Transglutaminase-synthesized γ-(glutamyl5) spermidine derivative of substance P is a selective tool for neurokinin-2 receptors characterization Peptides 19:683–690

  23. Mancuso F., Porta R., Calignano A., Di Pierro P., Sommella M.C., Esposito C., 2001 Substance P and its transglutaminase-synthesized derivatives elicit yawning behavior via nitric oxide in rats Peptides 22:1453–1457

  24. Mariniello L., Di Pierro P., Esposito C., Sorrentino A., Masi P., Porta R., 2003 Preparation and mechanical properties of edible pectin-soy flour films obtained in the absence or presence of transglutaminase Journal of Biotechnology 102:191–198

  25. Masárová J., Mislovičová D., Gemeiner P., Michalková E., 2001 Stability enhancement of Escherichia coli penicillin G acylase by glycosylation with yeast mannan Biotechnology and Applied Biochemistry 34:127–133

  26. Metafora S., Peluso G., Persico P., Ravagnan G., Esposito C., Porta R., 1989 Immunosuppresive and anti-inflammatory properties of a major protein secreted from the epithelium of the rat seminal vesicles Biochemical Pharmacology 38:121–131

  27. Mizuno A., Mitsuiki M., Motoki M., Ebisawa K., Suzuki E., 2000 Relationship between the glass transition of soy protein and molecular structure Journal of Agricultural and Food Chemistry 48:3292–3297

  28. Moskvichyov B.V., Komarov E.V., Ivanova G.P., 1986 Study of trypsin thermodenaturation process Enzyme and Microbial Technology 8:498–502

  29. Ohtsuka T., Sawa A., Kawabata R., Nio N., Motoki M., 2000 Substrate specificities of microbial transglutaminase for primary amines Journal of Agricultural and Food Chemistry 48:6230–6233

  30. Persico P., Calignano A., Mancuso F., Marino G., Pucci P., Esposito C., Mariniello L., Porta R., 1992 Substance P inactivation by transglutaminase in vitro Peptides 13:151–154

  31. Porta R., Esposito C., Gentile V., Mariniello L., Peluso G., Metafora S., 1990. Transglutaminase-catalysed modifications of SV-IV, a major protein secreted from the rat seminal vesicle epithelium International Journal of Peptide and Protein Research 35:117–122

  32. Ramírez H.L., Chico B., Hoste K., Schacht E.H., Villalonga R., 2002 Invertase stabilization by chemical modification of sugar chains with carboxymethylcellulose Journal of Bioactive and Compatible Polymers 17:161–172

  33. Schwert G.B., Takenaka Y., 1955 A spectrophotometric determination of trypsin and chymotrypsin Biochimica et Biophysica Acta. 16:570–575

  34. Srivastava R.A.K., 1991 Studies on stabilization of amylase by covalent coupling to soluble polysaccharides Enzyme and Microbial Technology 13:164–170

  35. Sundaram P.V., Venkatesh R., 1998 Retardation of thermal and urea induced inactivation of α-chymotrypsin by modification with carbohydrate polymers Protein Engineering 11:691–698

  36. Tricot M., 1984 Comparison of experimental and theoretical persistence length of some polyelectrolytes at various ionic strengths Macromolecules 17:1698–1704

  37. Tufano M.A., Porta R., Farzati B., Di Pierro P., Rossano F., Catalanotti P., Baroni A., Metafora S., 1996 Rat seminal vesicle protein SV-IV and its transglutaminase-synthesized polyaminated derivative SPD2-SV-IV induce cytokine release from human resting lymphocytes and monocytes in vitro Cellular Immunology 168:148–157

  38. Venkatesh R., Sundaram P.V., 1998 Modulation of stability properties of bovine trypsin after in vitro structural changes with a variety of chemical modifiers Protein Engineering 11:691–698

  39. Villalonga M.L., Fernández M., Fragoso A., Cao R., Villalonga R., 2003a Functional stabilization of trypsin by conjugation with β-cyclodextrin-modified carboxymethylcellulose Preparative Biochemistry and Biotechnology 33:53–66

  40. Villalonga R., Fernández M., Fragoso A., Cao R., Di Pierro P., Mariniello L., Porta R., 2003b Transglutaminase-catalysed synthesis of trypsin-cyclodextrin conjugates. Kinetics and stability properties Biotechnology and Bioengineering 81:732–737

  41. Villalonga R., Fernández M., Fragoso A., Cao R., Mariniello L., Porta R., 2003c Thermal stabilization of trypsin by enzymatic modification with β-cyclodextrin derivatives Biotechnology and Applied Biochemistry 38:53–59

  42. Villalonga R., Gómez L., Ramírez H.L., Villalonga M.L., 1999 Stabilization of α-amylase by chemical modification with carboxymethylcellulose Journal of Chemical Technology and Biotechnology 74:635–638

  43. Villalonga R., Villalonga M.L., Gómez L., 2000 Preparation and functional properties of trypsin modified by carboxymethylcellulose Journal of Molecular Catalysis B Enzymatic 10:483–490

  44. Walsh K.A., 1970 Trypsinogen and trypsin of various species Methods in Enzymology 19:41–63

  45. Wang, Ch., Eufemi M., Turano C., Giartosio A., 1996 Influence of the carbohydrate moiety on the stability of glycoproteins Biochemistry 35:7299–7307

  46. Washizu K., Ando K., Koikeda S., Hirose S., Matsuura A., Takagi H., Motoki M., Takeuchi K., 1994 Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans Bioscience Biotechnology and Biochemistry 58:82–87

Download references


This research was supported by the International Foundation for Science, Stockholm, Sweden, and the Organization for the Prohibition of Chemical Weapons, The Hague, The Netherlands, through a grant to R. Villalonga (Grant F/3004-1). Financial support from The Third World Academy of Sciences, through a grant to R. Villalonga (Grant 01-279 RG/CHE/LA), is also gratefully acknowledged.

Author information

Correspondence to Reynaldo Villalonga.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Villalonga, M.L., Villalonga, R., Mariniello, L. et al. Transglutaminase-catalysed glycosidation of trypsin with aminated polysaccharides. World J Microbiol Biotechnol 22, 595–602 (2006). https://doi.org/10.1007/s11274-005-9076-2

Download citation


  • Enzyme thermostability
  • modified enzyme
  • polysaccharide
  • transglutaminase
  • trypsin