Expression and enzymatic properties of a unique recombinant anticoagulant and fibrinolytic enzyme from Acinetobacter baumannii TU04

• Main Authors: Krishnan, Renuka, Chun, Shiong Chong, Kian, Mau Goh, Abdul Wahab, Firdaus, Jamaluddin, Haryati
• Format: Article
• Published: International Journal of Pharmacy and Pharmaceutical Science 2015
• Subjects: R Medicine (General)
• Online Access: http://eprints.utm.my/id/eprint/55204/

Objective: The objective of this research is to clone and express a new fibrinolytic enzyme encoding serine protease gene in Escherichia coli thus, characterize such purified recombinant. Methods: The recombinant clone was successfully expressed in Lemo21 system and purified using immobilized nickel cation affinity chromatography on a His•bind resin®, followed by ammonium sulfate precipitation and protein filtration in combination. General properties of the purified enzyme were investigated, including the molecular weight, effects of inhibitors and metal ions, substrate specificity, amylolytic activity, fibrinolytic activity and effect of anticoagulant activity in-vitro. Results: The recombinant clone was expressed in Lemo21 system in the cytoplasm in a soluble and active form. The resulting enzyme, SERpro was purified to homogeneity with a purification of 19.35-fold and recovery yield of 4.85%. The enzyme exhibited maximal activity at 37 °C and at pH7.4, respectively. The molecular weight of the purified enzyme was 82 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fibrinogenolysis peptide sequence analysis revealed that SERpro degraded Bß chain of Fibrin at a much lower rate but cleaved Aa and ?-chain extensively. The enzyme was activated by metal ions such as Mg2+, Fe3+and Zn2+, and was inhibited strongly by PMSF. The clotting time of human blood serum in the presence of 1U SERpro reached a relative partial thromboplastin time of 13.9% with a 1.14-fold increase. Conclusion: The study deduced SERpro as a new protease with anti-thrombotic activity from Acinetobacter baumannii TU04