Browsing by Author "Krishnan, Renuka P."

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    Molecular cloning and characterization of a new fibrinolytic enzyme from acinetobacter baumannII TU04
    (Universiti Teknologi Malaysia, 2016) Krishnan, Renuka P.
    In Malaysia, the occurrence of cardiovascular diseases has increased for the past thirty years and has remained as the number one killer. Fibrinolytic enzymes play a vital role in treating this disorder. However, their high production cost and undesirable side-effects circumscribe their widespread commercial use. This study aimed to clone, over-express, purify and characterize a new microbial fibrinolytic protease from Asian traditional fermented foods. A potent subtilisin-like serine protease gene encoding fibrinolytic enzyme from a newly isolated Acinetobacter baumannii TU04 was successfully cloned and expressed. The nucleotide sequence of the cloned gene revealed a single open reading frame of 2,184 bp coding for 736 amino acids and the deposited GenBank accession number is KP204011. The recombinant clone was expressed in the cytoplasm of E. coli Lemo 21 (DE3) as soluble and active enzyme. The resulting enzyme, SERpro was successfully purified via an immobilized nickel cation affinity chromatography column. SERpro was purified to homogeneity with a purification factor of 18-fold and recovery yield of 5%. SERpro exhibited maximal activity at 37 ºC and at pH 7.4, respectively. The molecular weight of the purified SERpro was about 82 kDa as 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 clotting time of human blood serum i.e; relative partial thromboplastin time increased by 1.14-fold increase (13.9 %) in the presence of 1U SERpro. SERpro exhibited analogous sequence similarity with other established fibrinolytic enzymes. As a conclusion, data suggests that SERpro from Acinetobacter baumannii TU04 is a potent protease with anti-thrombotic activity