EXPRESSION AND ENZYMATIC PROPERTIES OF A UNIQUE RECOMBINANT ANTICOAGULANT AND FIBRINOLYTIC ENZYME FROM ACINETOBACTER BAUMANNII TU04
Keywords:Fibrinolytic enzyme, Acinetobacter baumannii, subtilisin-like serine protease, Lemo21 expression host, Anticoagulant
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 Hisâ€¢bind resinÂ®, 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 Â°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 BÎ² chain of Fibrin at a much lower rate but cleaved AÎ± and Î³-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.
Kotb E. Fibrinolytic bacterial enzymes with thrombolytic activity. Springer Briefs Microbiol; 2012.
World Health Organization the world Health report; 2013.
Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 2006;3:442.
Hua Y, Jiang B, Mine Y, Mu WM. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste. J Agric Food Chem 2008;56:1451-7.
Peng Y, Yang XJ, Zhang YZ. Microbial fibrinolytic enzymes: an overview of source, production, properties and thrombolytic activity in vivo. Appl Microbiol Biotechnol 2005;69:126-32.
Wong AHK, Mine Y. Novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning. J Agric Food Chem 2004;52:980-6.
Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, et al. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK11-4 screened from Chungkook-Jang. Appl Environ Microbiol 1996;62:2482â€“8.
Lu FX, Lu ZX, Bie XM, Yao ZY, Wang YF, Lu YP, et al. Purification and characterization of a novel anticoagulant and fibrinolytic enzyme produced by endophytic bacterium Paenibacillus polymyxa EJS-3. Thromb Res 2010;126:349-55.
Mihara H, Sumi H, Yoneta T, Mizumoto H, Ikeda R, Seiki M, et al. A novel fibrinolytic enzyme extracted from the earthworm Lumbricus rubellus. Jpn J Physiol 1991;41:461-72.
Markland FS. Snake venom fibrinogenolytic and fibrinolytic enzymes: An updated Inventory. Thromb Haemostasis 1998;97:668-74.
Ahn MY, Hahn BS, Ryu KS, Kim JV, Kim I, Kim YS. Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles Catharsius molossus. Thromb Res 2003;112:339-47.
Maurer HR. Biochemistry, Pharmacology and medical use bromelain. Cell Mol Life Sci 2001;58:1234-45.
Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S. Purification and characterization of a strong fibrinolytic enzyme (Nattokinase) in vegetable cheese natto, a popular soybean fermented food in Japan. Biochem Biophys Res Commun 1993;197:1340-7.
Mine Y, Wong AHK, Jiang B. Fibrinolytic enzymes in Asian fermented foods. Food Res Int 2005;38:243-50.
Peng Y, Huang Q, Zhang R, Zhang YZ. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol B 2003;134:45-52.
Cannon CP, McCabe CH, Gibson CM, Ghali M, Sequeira RF, McKendall GR, et al. TNK-Tissue plasminogen activator in acute myocardial infarction. Jpn Circ J 1997;95:351-6.
Collen D. Fibrin selective thrombolytic therapy for acute myocardial infarction. J Circ 1996;93:857-65.
Blasi F, Sidenius N. The Urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling. Front Membr Biochem 2010;584:1923-30.
Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 1987;43:1110-1.
Sharul Aida MS. Isolation, Screening and characterization of fibrinolytic enzyme producing bacteria from Tapai. Master Dissertation in Biotechnology, Universiti Teknologi Malaysia, Johor Malaysia; 2012.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory: New York; 1989.
Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952;40:346-51.
Lee SK, Ho BD, Jong KT, Bok LS. Purification and characterization of a fibrinolytic enzyme from Bacillus sp. KDO-13 isolated from soybean paste. J Microbiol Biotechnol 2001;11:845-52.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;5259:680â€“5.
Batomunkueva BP, Egorov NS. Isolation, Purification and resolution of the extracellular proteinase complex of Aspergillus ochraceous 513 with fibrinolytic and anticoagulant activities. J Microbiol 2001;70:519-22.
Strukova SM, Kogan AE, Tara AA, Aaviksaar AA. The antithrombin effect of the snake venom protein C activator. Vopr Med Khim 1989;35:115-9.
Zhou H, Zhang T, Yu D, Pi B, Yang Q. Genomic analysis of multidrug-resistant Acinetobacter baumannii strain MDR-ZJ06 widely spread in China. Antimicrob Agents Chemother 2011;55:4506-12.
Chang CT, Fan MH, Kuo FC, Sung HY. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J Agric Food Chem 2000;48:3210-6.
Choi NS, Chang KT, Maeng PJ, Kim SH. Cloning, expression and fibrinolytic properties of subtilisin DJ-4 gene from Bacillus sp. DJ-4. FEMS Microbiol Lett 2004;236:325â€“31.
Walker JB, Nesheim ME. The molecular weights, Mass distribution, Chain composition, and Structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. J Biol Chem 1999;274:5201-12.
Wesam AH, Esam K, Nadia MA, Yehia AE. Fibrinolysis and anticoagulant potential of a metalloprotease produced by Bacillus subtilis K42. J Biosci 2011;35:773-9.