Mutagenesis for Hyper Production of Streptokinase from Streptococcus Equisimilis and Comparative Sequence Analysis of SK Gene

Abstract

Streptokinase (SK) is protein excreted by strains of β-hemolytic Streptococci. It is applied as treatment of life-threatening diseases thrombosed arteriovenous shunts, and thromboembolism. Streptokinase is naturally produced by several bacterial strains especially Streptococci. In connection with its broad application in the field of medicine, there is an urgent need to search for methods of its hyper production. Present study aimed at SK hyper production by mutagenesis of S. equisimilis strain. The wild bacterial strain S. equisimilis was subjected to random mutagenesis (UV irradiation and Ethidium bromide) for enhanced streptokinase production. The modified strain was assessed for the activity of streptokinase. The mutant S. equisimilis UV6 showed the maximum SK production and activity. By standardizing the effects of different factors like substrate (CSL), carbon source, salt concentration, temperature and pH an optimum fermentation protocol for SK production was established. The higher SK production was observed in mutant strain UV6 601 U mL-1 with increased activity up to 400 %. The SK was purified and its kinetics data indicated improved activity as its Km value was low as compared to wild SK. The SK gene from mutant UV6 was amplified and cloned in E. coli using pTZ57R/T vector. The gene was sequenced and compared with the wild SK gene to check the genetic difference between wild and mutant strain. In our study, the comparative sequence analysis revealed 26 point mutations, among these 3 were transitions (A to G) while the remaining 23 were transversions (A/G to C/T) in coding region of sk gene. Moreover, 3 amino acid deletions at the C terminal region of SK protein were predicted due to non-sense mutation in coding region (resulting in a stop codon after residue 437). The activity of any protein is direcly linked with amino acid composision. in our studies, the in-silico analyses predicted that amino acid residues at position 22, 62, 64 and 80 could be accountable in SK hyperactivity.