EXPRESSION AND ENZYMATIC PROPERTIES OF A UNIQUE RECOMBINANT ANTICOAGULANT AND FIBRINOLYTIC ENZYME FROM ACINETOBACTER BAUMANNII TU04 Original Article RENUKA KRISHNAN, CHUN SHIONG CHONG, KIAN MAU GOH, FIRDAUS ABDUL WAHAB, HARYATI JAMALUDDIN* Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Email: haryati@fbb.utm.my Received: 04 Aug 2015 Revised and Accepted: 27 Oct 2015 ABSTRACT 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 Bβ chain of Fibrin at a much lower rate but cleaved Aα and γ-chain extensively. The enzyme was activated by metal ions such as Mg 2+ , Fe 3+ and Zn 2+ Conclusion: The study deduced SERpro as a new protease with anti-thrombotic activity from Acinetobacter baumannii TU04. , 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. Keywords: Fibrinolytic enzyme, Acinetobacter baumannii, subtilisin-like serine protease, Lemo21 expression host, Anticoagulant. INTRODUCTION Cardiovascular diseases (CVDs) are caused by the disorders of heart and blood vessels [1]. According to health data statistic published by World Health Organization (WHO) 2013, CVDs are projected to remain as the single leading cause of mortality, accounting for 23.3 million deaths in 2030 [2-3]. Thromboembolism is the most prevalent type of CVD [4]. Thrombolytic therapy is the best way to achieve recanalization for this complication [5-8]. Thrombolytic agents characterized by its ability to hydrolyze thrombi via direct degradation or plasmin activation reveals a significant importance in the prevention and treatment of thrombosis [8]. Various potent thrombolytic agents have been successfully identified and characterized from animals [9-11], plants [12] and microorganism [7, 13-15]. Recently, studies have also demonstrated the efficacy of thrombolytic agents such as tissue-type plasminogen (t-PA) [16-17], single-chain urokinase-type plasminogen activator (scu-PA or pro-urokinase, Pro-UK) [18] and plasmin-like protein [19] in the recanalization and reperfusion of thrombo-embolism. However, their hemorrhagic side-effects, low specificity, modest reliability and benefits [8] have prompted a quest for alternative agents, from other sources that are safer and more potent to be developed into therapeutic drugs for thrombo-embolism. In general, fibrinolytic agents derived from Asian traditional fermented foods can function as an adjunct to conventional cardiovascular disease therapy through their incorporation as food additives and nutraceutical application, which can help in preventing cardiovascular disease [6, 14]. In this study, a bacterial strain isolated from Tapai Ubi, a traditional Malaysian fermented food, identified as Acinetobacter baumanii TU04 [20] were analyzed for its thrombolytic potential. Prior to our report, fibrinolytic proteases had not yet been reported from Acinetobacter baumannii, therefore, in this study we describe the molecular cloning and expression of a new fibrinolytic enzyme encoding serine protease gene in Escherichia coli and the characterization of the purified recombinant enzyme. MATERIALS AND METHODS Chemicals Human plasma fibrinogen, Thrombin (EC 3.4.21.5) from bovine (Calbiochem Cat# 605157), Plasmin from human plasma (10 602 361 001) [5U], Plasminogen, Perfect Protein TM Bacterial strains and growth conditions Marker (Novagen, 69149-3) and Prestained Protein Marker, (NEB#P7702) were used. Specific kits for sample extraction and purification were purchased from Promega and Qiagen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) set and reagents were bought from Bio-Rad Co. (Richmond, CA, USA). Chromatography column and His® Bind resin were purchased from GE Healthcare. Other fine chemicals and reagents used were of analytical grade. Acinetobacter baumannii TU04 isolated from Tapai Ubi, a traditional cassava-fermented food, was grown at 37 °C, 200rpm for 16 h in basal medium containing 1% (w/v) tryptone, 0.5% (w/v) beef extract, 1% (w/v) NaCl, pH 7.0±0.2 (Accession No: KP204010). Escherichia coli DH5α was used in cloning steps and Lemo21 (DE3), which contains the T7 RNA polymerase gene under the IPTG-inducible lacUV5 promoter, was used for the expression of the enzyme under T7 promoter control. Escherichia coli strains were cultured in Luria-Bertani (LB) medium supplemented with ampicillin of 100 µg/ml at 37 °C when necessary. Construction of protease cloning and expression system Genomic DNA of Acinetobacter Baumannii TU04 was isolated by the procedure as described Sambrook (1989) [21]. Two degenerate oligonucleotide primers were synthesized to amplify DNA fragments encoding the subtilisin-like serine protease gene: forward primer, AsLfEco R1: 5’ GCC GCA GAA TTC ATG ACA AGG ATA ATC GTA GCA TCC 3’ (with Eco R1 restriction site as underlined) and reverse primer, AsLrXho 1: 5’ CTG GCT TCT CGA GCT AAA CAA CTG GGT AAG TCT GAT 3’ (with Xho 1 restriction site shown as underlined). These primers were synthesized based on sequences of the conserved International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 7, Issue 12, 2015 Innovare Academic Sciences