Technologies

   1. Technology for recombinant Serratiopeptidase production

• Production of Serratiopeptidase by engineered Escherichia coli strain for therapeutic application
  Background
  Serratiopeptidase is one of the important proteases which belong to the serine protease family. It is an extracellular zinc-containing metalloprotease that is produced by Serratia marcescens having molecular weight of about 53kD. It has shown therapeutic (anti-inflammatory, anti-fibrinolytic and analgesic) as well as industrial applications (detergents, food processing, leather, paper and brewing etc.). The evolution of Serratia marcescens as an opportunistic pathogen associated with various infections has led researchers to think and develop an alternate strategy for its industrial production. Serratiopeptidase is also taken as a supplement to augment the overall health of the cardiovascular system. It has also shown to reduce the cancer metastasis by thinning its extracellular matrix as well as to solubilize non-living tissues such as mucous, plaques and blood clots. It has the potential to cure and treat several disorders such as atherosclerosis, arthritis, bronchitis, carpal tunnel syndrome, fibrocystic breast disease, Crohn’s disease, leg ulcers, traumatic swelling, fibromyalgia, breast engorgement, migraine, Alzheimer’s disease, sinusitis, hepatitis, lung disorders, arthritis, diabetes, carotid artery blockage, thrombosis, uterine fibroids. Currently the industrial production of serratiopeptidase makes use of natural S. marcescens strain. However, many challenges and issues have arisen to use this strain because of pathogenic nature, lack of genetic tools, unclear mechanism of the utilization of cheaper carbon and nitrogen sources, industrial -scale fermentation and process. The bacteria is also reported to cause pneumonia, septicemia, and as well is associatedwith cystic fibrosis. Therefore, a pressing need hasarisen to find an alternative approach for the production of active and efficient serratiopeptidase
  Technology
  GBRC make successful cloning, expression and purification of active serratiopeptidase, using Escherichia coli BL21 [DE3] and pET SUMO vector. Further it also optimize synthetic media and culture conditions for enhanced serratiopeptidase production. Initial optimization of physical parameters was done followed by a screening of different carbon and nitrogen sources. The significant media components for serratiopeptidase production as shown by factorial screening experiment were subjected to Response Surface Methodology (RSM) based optimization.
  Potential Applications
  The success of the application of a statistical model for designing an optimized media for enhanced serratiopeptidase production also suggests a new insight for the scale up of serratiopeptidase towards industrial applications.
  Technology Status
  Technology is ready and the optimized media yielded 2.5 ± 0.764 g L-1 of protein was obtained having 8382±291 U mg-1 of specific caseinolytic activity.

  

   2. Technology for recombinant streptokinase production

• Higher production of Streptokinase in Streptococcus equisimilis by CRISPR-Cas9 Mediated Knockout of SagD gene
  Background
  Streptokinase is a thrombolytic enzyme with a molecular weight of 47 kDa that is spontaneously generated by the alpha hemolytic group of Streptococcus species. Streptokinase forms a 1:1 streptokinase:plasminogen complex, which is utilized in healthcare to break up blood clots (due to myocardial infractions) and save lives. Streptokinase is used in the treatment of myocardial infections, arteriovenous cannula occlusion, embolism, and deep vein thrombosis. It is worth USD 40 million on the open market. Streptokinase production in other host factors, such as Escherichia coli, has a problem with product toxicity or other factors, such as the absence of short regulatory RNAs in the expression host for the stability of streptokinase mRNA transcript. As a result, there is an urgent need to create natural producers of streptokinase.
  Technology
  We used CRISPR-Cas9 to successfully knockout the SagD gene from Streptococcus equisimilis and observed a 13.58-fold increased expression of streptokinase at the transcript level and 1.48-fold higher expression at the protein level in the mutant strain compared to wild type.
  Potential Applications
  Our CRISPR-Cas9-based approach can be used in other microorganisms for genome editing for other industrially important therapeutic production.
  Technology Status
  Technology is ready and already tested

  

Expression of Interest for technologies developed by GBRC