Enhanced Production Of Streptokinase By Chemical Mutagenesis Of Streptococcus agalactiae EBL-20

Abstract

Streptokinase (SK) is an enzyme that is used for the treatment of cardiovascular diseases. The current study focused on the enhanced production of SK by inducing mutation in Streptococcus agalactiae EBL-20 and optimization of medium components and culture conditions for the maximum growth of mutant derived strain. S. agalactiae EBL-32 was selected as a potent mutant after exposure of S. agalactiae EBL-20 to EMS for 180 minutes. SK activity obtained from mutant derived strain was found to be 1.6 fold higher as compared to the activity achieved by wild strain. Nutritional requirements of the mutated strain were optimized by single factor analysis method suggesting glucose as the optimum carbon source; yeast extract and peptone as a suitable nitrogen sources and corn steep liquor (CSL) as an appropriate substrate for the maximum SK production. The culture conditions determined by response surface methodology (RSM) suggested that a temperature value of 37.5⁰C and pH 7 of the fermentation medium with 2.50 mL inoculum size for 36 hours of incubation was optimum for maximum yield of SK. Hence the optimization studies resulted HIGHLIGHTS  Streptokinase isolated from Streptococcus agalactiae is an active thrombolytic agent  Ethyl methanesulfonate is an effective mutagenic agent for Streptococcus spp.  Response surface methodology resulted in 1.92-fold increased streptokinase activity Arshad, A.; et al. Brazilian Archives of Biology and Technology. Vol.62: e19170813, 2019-www.scielo.br/babt into 1.92 fold increase in the yield of SK suggesting the new isolate suitable for commercial scale production of SK.

CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

“…Some have used Streptococcus agalactiae with nonspecific ethyl methanesulfonate (EMS) based chemical mutagenesis [44], Streptococcus sanguis produced lowered molecular mass containing streptokinase [45], B. sublitis with C-terminally processed streptokinase [46]. Relative comparison of streptokinase activity in different hosts was as follows, Saccharomyces pombe (2450 IU), Streptococcus equisimilis (100-150 IU), Pichia pastoris (3200 IU) and E. coli (1000-1500 IU) [44,[47][48][49]. All these studies observed that streptokinase degradation of matured streptokinase 47kDa to inactivated 44kDa [45,46,50].…”

“…Studies on the expression of streptokinase with different expression vectors/strategies and the host are available. Some have used Streptococcus agalactiae with nonspecific ethyl methanesulfonate (EMS) based chemical mutagenesis [44], Streptococcus sanguis produced lowered molecular mass containing streptokinase [45], B. sublitis with C-terminally processed streptokinase [46]. Relative comparison of streptokinase activity in different hosts was as follows, Saccharomyces pombe (2450 IU), Streptococcus equisimilis (100-150 IU), Pichia pastoris (3200 IU) and E. coli (1000-1500 IU) [44,[47][48][49].…”

Production and market comparison of urokinase and streptokinase as effective and cheap fibrinolytic agents for treatment of cardiovascular diseases

“…Next, by inducing the expression vector, high expression of the recombinant protein recSK was produced which held little or no capacity to react as an immunogen (Deepak & Geetha, 2019). Another study carried out by (Arshad, Zia, Asghar, & Joyia, 2019) in an attempt to achieve the said objective, enhanced production of streptokinase was obtained with little immunogenicity by chemical mutagenesis of S. agalactiae.…”

“…The microbial synthesis of streptokinase has been further reported to increase using mutant microbial species and optimization of fermentation medium. Arshad et al, (2019) reported a 4.14-fold increase in streptokinase production by optimization of fermentation media. Similarly, Wu et al, (2019) developed a cheap fermentation media for the cost-effective synthesis of streptokinase.…”

“…The challenge for cost-effective production hence remains valid (Vimal & Kumar, 2019). The market potential of thrombolytic therapy using drugs like streptokinase and urokinase has been divided into segments, classified based on drug type, application, distribution channel, and region (Arshad et al, 2019) (Table 3).…”