July-August 2021 Indian Journal of Pharmaceutical Sciences 856 Research Paper Isolation, Identification and Sequence Analysis of Subtilisin Gene (Quaking Homolog) Encoding a Fibrinolytic Enzyme from Bacillus subtilis Z. MUSHTAQ, S. AHMED 1 , FATEHA IRSHAD AND G. MUSTAFA 2 * Bioactive Molecules Research Lab, Department of Biochemistry, University of Agriculture, Faisalabad, Punjab 38000, Pakistan, 1 Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive La Jolla, California 92093, United States of America, 2 Department of Biochemistry, Government College University, Faisalabad 38060, Pakistan Mushtaq et al.: Characterization of Subtilisin Gene from Bacillus subtilis Fibrinolytic therapy progressed by the evolution of diferent strategies that helped in enhancing its efcacy and specifcity. The use of microbial fbrinolytic enzymes is leading to a promising direction for the treatment of cardiovascular diseases. Subtilisin, a member of subtilases is a fbrinolytic enzyme abundantly found in Bacillus species. The isolation of subtilisin gene (quaking homolog) from Bacillus subtilis was attempted in the present work. The genomic deoxyribonucleic acid extraction was done following Yamada protocol and used as a template for polymerase chain reaction amplifcation of the target gene using specifcally designed primers. The polymerase chain reaction product was ligated into cloning vector pTZ57R/T followed by its transformation into Escherichia coli top 10 strain. A 1090 base pair, partial gene sequence was amplifed coding for subtilisin protein of 363 amino acids with molecular weight of 37550.7 Daltons. The nucleotide sequence revealed signifcant evolutionary relationships with subtilases from other strains of Bacillus subtilis. Our study confrms the presence of subtilisin (quaking homolog) gene in local Bacillus species suggesting economical way to produce important thrombolytic agents of commercial and pharmaceutical worth. Key words: Fibrinolytic enzymes, subtilisin, Bacillus subtilis, vector pTZ57R/T, thrombolytic agentst Cardiovascular diseases (CVDs) have become the leading cause of mortality throughout the world [1] , including acute myocardial infarction, peripheral vascular disease, ischemic heart disease, high blood pressure, arrhythmias and stroke. Myocardial infarction and CVDs stem from the thrombosis in the coronary artery leading to fatal medical complications [2] . Various factors contribute to the thrombus formation in blood vessels however, fbrin is the main component of the blood clot which is produced after trauma or injury from fbrinogen by the action of thrombin [3] . Anticoagulants such as heparin and coumarin were used for the treatment of thrombosis in earlier days which have been substituted by the enzyme-based therapies involving in vivo lysis of fbrin [1] . The use of thrombolytic agents including streptokinase, urokinase and tissue-plasminogen activators for thrombosis confronted some limitations that confne their usage such as bleeding complications, resistance to reperfusion, incidence of acute coronary reocclusion and the high cost [2,4] . Research for improved thrombolytic agents with enhanced efcacy is being pursued involving the construction of genetically modifed plasminogen activators and bacterial fbrinolytic enzymes. In countries like China, Korea, India, Japan and West Africa, soybean-fermented foods had been used as a staple food for a long time. The signifcance of these foods lies in their potential of being thrombolytic that helps in preventing diseases like heart attacks, senility, strokes and osteoporosis [5] . These enzymes also have potential to be used in food fortifcation and nutraceutical applications as they can prevent CVDs [6] . *Address for correspondence E-mail: gmustafa_uaf@yahoo.com Accepted 21 August 2021 Revised 01 January 2021 Received 08 October 2019 Indian J Pharm Sci 2021;83(4):856-864 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms