A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization

• Published in: PloS one 2020-10, Vol.15 (10), p.e0240920-e0240920
• Main Authors: Gao, Li-Wei, Zhu, Hong-Tao, Liu, Cai-Yun, Lv, Zhi-Xiang, Fan, Xiao-Man, Zhang, Ye-Wang
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Analysis; Anticoagulants; Bacterial proteins; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteroides; Bacteroides - enzymology; Bacteroides - genetics; Binding Sites; Biology and Life Sciences; By-products; Calcium; Calcium - metabolism; Calcium ions; Carbon dioxide; Chromatography; Cloning; Cloning, Molecular; Cobalt; Deoxyribonucleic acid; DNA; E coli; Enzyme Activation; Enzymes; Ethylenediaminetetraacetic acids; Glycosaminoglycans; Heparan sulfate; Heparin; Heparin - metabolism; Heparin Lyase - chemistry; Heparin Lyase - genetics; Heparin Lyase - metabolism; Homology; Hydrogen; Hydrogen Bonding; Hydrogen bonds; Industrial production; Low molecular weights; Magnesium; Magnesium - metabolism; Manganese - metabolism; Medicine and Health Sciences; Models, Molecular; Molecular Docking Simulation; Molecular weight; Mucopolysaccharides; Pharmacy; Physical Sciences; Plasmids; Protein Binding; Protein Conformation; Proteins; Research and Analysis Methods; Sodium; Structure; Substrates; Zinc
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0240920

As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its' important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca2+, Mg2+, and Mn2+, while severely inhibited by Zn2+, Co2+, and EDTA. The specific activity of the enzyme was 738.3 U·mg-1 which is the highest activity ever reported. The Km and Vmax were calculated as 0.17 mg·mL-1 and 740.58 U·mg-1, respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.