Annals of Microbiology

, Volume 68, Issue 6, pp 409–418 | Cite as

Deciphering the mode of action, structural and biochemical analysis of heparinase II/III (PsPL12a) a new member of family 12 polysaccharide lyase from Pseudopedobacter saltans

  • Karthika Balasubramaniam
  • Kedar Sharma
  • Aruna Rani
  • Vikky Rajulapati
  • Arun Goyal
Original Article


Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K m and Vmax, for PsPL12a were 4.6 ± 0.5 mg/ml and 70 ± 2 U/mg, respectively. Ten millimolars of each Mg2+ and Mn2+ ions enhanced PsPL12a activity by 80%, whereas Ni2+ inhibited by 75% and Co2+ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg2+ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.


Glycosaminoglycans Heparin Heparinase Pseudopedobacter saltans 



The authors thank DBT Program Support, IIT Guwahati, for CD analysis and Central Instrumentation Facility for ESI-mass analysis. Fellowship provided by the Ministry of Human Resource Development, Govt. of India, to Karthika B. is gratefully acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Amin K (2012) The role of mast cells in allergic inflammation. Respir Med 106(1):9–14CrossRefPubMedGoogle Scholar
  2. Bellamy RW, Horikoshi K (1992) U.S. patent no. 5,145,778. U.S. Patent and Trademark Office, Washington, DCGoogle Scholar
  3. Böhmer LH, Pitout MJ, Steyn PL, Visser L (1990) Purification and characterization of a novel heparinase. J Biol Chem 265(23):13609–13617PubMedGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254CrossRefPubMedGoogle Scholar
  5. Cao J, Lai Q, Li G, Shao Z (2014) Pseudopedobacter beijingensis gen. nov., sp. nov., isolated from coking wastewater activated sludge, and reclassification of Pedobacter saltans as Pseudopedobacter saltans comb. nov. Int J Syst Evol Microbiol 64(6):1853–1858CrossRefPubMedGoogle Scholar
  6. Chen Y, Maguire T, Hileman RE, Fromm JR, Esko JD, Linhardt RJ, Marks RM (1997) Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat Med 3(8):866–871CrossRefPubMedGoogle Scholar
  7. Chen S, Ye F, Chen Y, Chen Y, Zhao H, Yatsunami R, Nakamura S, Arisaka F, Xing XH (2011) Biochemical analysis and kinetic modeling of the thermal inactivation of MBP-fused heparinase I: implications for a comprehensive thermostabilization strategy. Biotechnol Bioeng 108(8):1841–1851CrossRefPubMedGoogle Scholar
  8. Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3(9):853–859CrossRefPubMedGoogle Scholar
  9. Desai UR, Wang HM, Linhardt RJ (1993) Specificity studies on the heparin lyases from Flavobacterium heparinum. Biochemistry 32(32):8140–8145CrossRefPubMedGoogle Scholar
  10. Doneanu CE, Chen W, Gebler JC (2009) Analysis of oligosaccharides derived from heparin by ion-pair reversed-phase chromatography/mass spectrometry. Anal Chem 81(9):3485–3499CrossRefPubMedGoogle Scholar
  11. Dreyfuss JL, Regatieri CV, Jarrouge TR, Cavalheiro RP, Sampaio LO, Nader HB (2009) Heparan sulfate proteoglycans: structure, protein interactions and cell signaling. An Acad Bras Cienc 81(3):409–429CrossRefPubMedGoogle Scholar
  12. Ernst S, Langer R, Cooney CL, Sasisekharan R (1995) Enzymatic degradation of glycosaminogIycans. Crit Rev Biochem Mol Biol 30(5):387–444CrossRefPubMedGoogle Scholar
  13. Esko JD, Selleck SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem 71(1):435–471CrossRefPubMedGoogle Scholar
  14. Gallagher JT, Turnbull JE (1992) Heparan sulphate in the binding and activation of basic fibroblast growth factor. Glycobiology 2(6):523–528CrossRefPubMedGoogle Scholar
  15. Garron ML, Cygler M (2010) Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology 20(12):1547–1573CrossRefPubMedGoogle Scholar
  16. Gasimli L, Robert JL, Dordick JS (2012) Proteoglycans in stem cells. Biotechnol Appl Biochem 59(2):65–76CrossRefPubMedGoogle Scholar
  17. Gray E, Hogwood J, Mulloy B (2012) The anticoagulant and antithrombotic mechanisms of heparin. In: Heparin-A century of Progress. Springer, Berlin Heidelberg, pp 43–61CrossRefGoogle Scholar
  18. Hovingh P, Linker A (1970) The enzymatic degradation of heparin and heparitin sulfate III. Purification of a heparitinase and a heparinase from flavobacteria. J Biol Chem 245(22):6170–6175PubMedGoogle Scholar
  19. Hulett MD, Hornby JR, Ohms SJ, Zuegg J, Freeman C, Gready JE, Parish CR (2000) Identification of active-site residues of the pro-metastatic endoglycosidase heparanase. Biochemistry 39(51):15659–15667CrossRefPubMedGoogle Scholar
  20. Hyun YJ, Lee JH, Kim DH (2010) Cloning, overexpression, and characterization of recombinant heparinase III from Bacteroides stercoris HJ-15. Appl Microbiol Biotechnol 86(3):879–890CrossRefPubMedGoogle Scholar
  21. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874CrossRefPubMedGoogle Scholar
  22. Limtiaco JF, Beni S, Jones CJ, Langeslay DJ, Larive CK (2011) NMR methods to monitor the enzymatic depolymerization of heparin. Anal Bioanal Chem 399(2):593–603CrossRefPubMedGoogle Scholar
  23. Lindahl U, Lidholt K, Spillmann D, Kjellén L (1994) More to “heparin” than anticoagulation. Thromb Res 75(1):1–32CrossRefPubMedGoogle Scholar
  24. Linhardt RJ, Fitzgerald GL, Cooney CL, Langer R (1982) Mode of action of heparin lyase on heparin. Biochim Biophys Acta Protein Struct Mol Enzymol 702(2):197–203CrossRefGoogle Scholar
  25. Linhardt RJ, Turnbull JE, Wang HM, Loganathan D, Gallagher JT (1990) Examination of the substrate specificity of heparin and heparan sulfate lyases. Biochemistry 29(10):2611–2617CrossRefPubMedGoogle Scholar
  26. Lohse DL, Linhardt RJ (1992) Purification and characterization of heparin lyases from Flavobacterium heparinum. J Biol Chem 267(34):24347–24355PubMedGoogle Scholar
  27. Lundin L, Larsson H, Kreuger J, Kanda S, Lindahl U, Salmivirta M, Claesson-Welsh L (2000) Selectively desulfated heparin inhibits fibroblast growth factor-induced mitogenicity and angiogenesis. J Biol Chem 275(32):24653–24660CrossRefPubMedGoogle Scholar
  28. Nader HB, Porcionatto MA, Tersariol IL, Pinhal MA, Oliveira FW, Moraes CT, Dietrich CP (1990) Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy. J Biol Chem 265(28):16807–16813PubMedGoogle Scholar
  29. Olsson P, Sanchez J, Mollnes TE, Riesenfeld J (2000) On the blood compatibility of end-point immobilized heparin. J Biomater Sci Polym Ed 11(11):1261–1273CrossRefPubMedGoogle Scholar
  30. Rabenstein DL (2002) Heparin and heparan sulfate: structure and function. Nat Prod Rep 19(3):312–331CrossRefPubMedGoogle Scholar
  31. Saad OM, Ebel H, Uchimura K, Rosen SD, Bertozzi CR, Leary JA (2005) Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase. Glycobiology 15(8):818–826CrossRefPubMedGoogle Scholar
  32. Sampaio LO, Tersariol IL, Lopes CC, Bouças RI, Nascimento FD, Rocha HA, Nader HB (2006) Heparins and heparans sulfates. Structure, distribution and protein interactions. Insights into Carbohydrate Structure and Bological Function, 1–24Google Scholar
  33. Sarrazin S, Lamanna WC, Esko JD (2011) Heparan sulfate proteoglycans. Cold Spring Harb Perspect Biol 3(7):a004952CrossRefPubMedPubMedCentralGoogle Scholar
  34. Shively JE, Conrad HE (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15(18):3932–3942CrossRefPubMedGoogle Scholar
  35. Shriver Z, Capila I, Venkataraman G, Sasisekharan R (2012) Heparin and heparan sulfate: analyzing structure and microheterogeneity. Heparin-A Century of Progress. Springer Berlin Heidelberg. 159–176Google Scholar
  36. Steyn PL, Segers P, Vancanneyt M, Sandra P, Kersters K, Joubert JJ (1998) Classification of heparinolytic bacteria into a new genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. proposal of the family Sphingobacteriaceae fam. nov. Int J Syst Evol Microbiol 48(1):165–177Google Scholar
  37. Thanawiroon C, Rice KG, Toida T, Linhardt RJ (2004) Liquid chromatography/mass spectrometry sequencing approach for highly sulfated heparin-derived oligosaccharides. J Biol Chem 279(4):2608–2615CrossRefPubMedGoogle Scholar
  38. Toyoshima M, Nakajima M (1999) Human heparanase purification, characterization, cloning, and expression. J Biol Chem 274(34):24153–24160CrossRefPubMedGoogle Scholar
  39. Xiao Z, Zhao W, Yang B, Zhang Z, Guan H, Linhardt RJ (2010) Heparinase 1 selectivity for the 3, 6-di-O-sulfo-2-deoxy-2-sulfamido-α-D-glucopyranose (1, 4) 2-O-sulfo-α-L-idopyranosyluronic acid (GlcNS3S6S-IdoA2S) linkages. Glycobiology 21(1):13–22CrossRefPubMedPubMedCentralGoogle Scholar
  40. Yamada S, Sugahara K (2003) Preparation of oligosaccharides from sulfated glycosaminoglycans using bacterial enzymes. In: Capillary electrophoresis of carbohydrates, pp 71–78CrossRefGoogle Scholar
  41. Yates EA, Rudd TR (2016) Recent innovations in the structural analysis of heparin. Int J Cardiol 212:S5–S9CrossRefPubMedGoogle Scholar
  42. Yoshida E, Sakai K, Tokuyama S, Miyazono H, Maruyama H, Morikawa K, Tahara Y (2002) Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans. Biosci Biotechnol Biochem 66(5):1181–1184CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Authors and Affiliations

  • Karthika Balasubramaniam
    • 1
  • Kedar Sharma
    • 1
  • Aruna Rani
    • 1
  • Vikky Rajulapati
    • 1
  • Arun Goyal
    • 1
  1. 1.Carbohydrate Enzyme Biotechnology Laboratory, Department of Biosciences & BioengineeringIndian Institute of Technology GuwahatiGuwahatiIndia

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