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
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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.
KeywordsGlycosaminoglycans 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.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Bellamy RW, Horikoshi K (1992) U.S. patent no. 5,145,778. U.S. Patent and Trademark Office, Washington, DCGoogle Scholar
- 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
- 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
- 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
- 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
- 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