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Isolation, Identification and Sequence Analysis of Subtilisin Gene (Quaking Homolog) Encoding a Fibrinolytic Enzyme from Bacillus subtilis

Author(s): Z. Mushtaq, S. Ahmed, Fateha Irshad and G. Mustafa*
Bioactive Molecules Research Lab, Department of Biochemistry, University of Agriculture, Faisalabad, Punjab 38000, Pakistan, 1Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive La Jolla, California 92093, United States of America, 2Department of Biochemistry, Government College University, Faisalabad 38060, Pakistan

Correspondence Address:
G. Mustafa, Department of Biochemistry, Government College University, Faisalabad 38060, Pakistan, E-mail:

Fibrinolytic therapy progressed by the evolution of different strategies that helped in enhancing its efficacy and specificity. The use of microbial fibrinolytic enzymes is leading to a promising direction for the treatment of cardiovascular diseases. Subtilisin, a member of subtilases is a fibrinolytic 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 amplification of the target gene using specifically 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 amplified coding for subtilisin protein of 363 amino acids with molecular weight of 37550.7 Daltons. The nucleotide sequence revealed significant evolutionary relationships with subtilases from other strains of Bacillus subtilis. Our study confirms the presence of subtilisin (quaking homolog) gene in local Bacillus species suggesting economical way to produce important thrombolytic agents of commercial and pharmaceutical worth.

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