title: Exploring the In Vitro Thrombolytic Activity of Nattokinase From a New Strain Pseudomonas aeruginosa CMSS.
abstract: BACKGROUND
  Thrombolytic therapy has become a conventional treatment for acute myocardial infarction (AMI), yet currently, clinically prescribed thrombolytic drugs have problems such as delayed action and other side effects. Fibrinolytic enzymes have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process, including plasmin activation. Nattokinase (NK) is a potent fibrinolytic agent for thrombosis therapy.
  
  
  OBJECTIVE
  The aim of this study was to enhance the production of NK from Pseudomonas aeruginosa CMSS by media optimization and strain improvement.
  
  
  METHODS
  In the present study, a potent NK-producing strain was isolated from cow milk and identified. To enhance the yield of NK, effect of various parameters such as pH, temperature, carbon source, nitrogen source and inoculum size were optimized. Strain improvement of P. aeruginosa CMSS was done by random UV-mutagenesis. Nattokinase was partially purified and the activity was determined by the casein digestion method, blood clot lysis and fibrin degradation assay.
  
  
  RESULTS
  Based on morphological, biochemical and molecular characterization, the strain was confirmed as P. aeruginosa (GenBank accession number: JX112657), designated as P. aeruginosa CMSS. The optimum condition at pH 7 and temperature at 25˚C showed activity of NK as 1514 U mL(-1) and 1532 U mL(-1), respectively. Sucrose as the carbon source and shrimp shell powder (SSP) as the nitrogen source expressed NK activity of 1721 U mL(-1) and 2524 U mL(-1), respectively. At 1% inoculum size, the maximum rate of enzyme production was achieved with 2581 U mL(-1). The NK activity of the mutant strain UV60 was 4263 U mL(-1), indicating a two-fold increase in activity compared to the wild strain (2581 UmL(-1)). Nattokinase produced from mutant strain P. aeruginosa CMSS UV60 showed 94% blood clot lysis at ten minutes. The degradation of fibrin clot by the produced NK was observed after two hours of incubation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the molecular mass of CMSS UV60 NK to be 21kDa.
  
  
  CONCLUSIONS
  The current study demonstrated the enhanced production of NK by P. aeruginosa CMSS. This study is unique and the findings are the first report on the production of NK from P. aeruginosa CMSS isolated from cow milk.
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Subathra Devi
authors.initials: SD
authors.lastname: Chandrasekaran
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Mohanasrinivasan
authors.initials: M
authors.lastname: Vaithilingam
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Ravi
authors.initials: R
authors.lastname: Shanker
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Swathi
authors.initials: S
authors.lastname: Thiyur
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Vaishnavi
authors.initials: V
authors.lastname: Babu
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Jemimah Naine
authors.initials: JN
authors.lastname: Selvakumar
authors.affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Suyash
authors.initials: S
authors.lastname: Prakash
keywords: Blood; Clot Busters; Nattokinase; Therapeutics; Thrombus