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.Objectives:The aim of this study was to enhance the production of NK from Pseudomonas aeruginosa CMSS by media optimization and strain improvement.Materials and 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.

Enhanced Production of Fibrinolytic Enzyme from Pseudomonas aeruginosa by Optimization Media Components

“…When using glucose, while the lowest specific efficacy was 14 U/mg when using glycerol (Figure 1). The current study does not agree with [19] as the best carbon source was sucrose, but the least effective carbon source is glycerol. The difference in results depends on the conditions and location of the bacterial isolation.…”

“…The results showed that the best inoculum size of the bacterial was 1% and the highest specific efficacy reached to 129.5 U/mg at a concentration of 1%, while the lowest specific efficacy was 47.4 U/mg when using 2.5 (Figure 4) .This study agreed with [19] where the best size of the inoculum was 1%, noting that the specific effectiveness of the enzyme decreases when increasing the concentration of the inoculum size.…”

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease

“…[42]. Sixteen NK gene sequences have been identified from various B. subtilis strains and their sequences are available in the NCBI GenBank.…”

Substantial improvement of protease production using statistical optimization and Downstream approach from the strain Bacillus toyonensis VKB5 sp., isolated from the host Actinidia delicosa

“…A standard graph of tyrosine was used to estimate the protease activity. A single unit of protease activity (U) was defined as the amount of the enzyme that releases one µM of tyrosine equivalent per ml of the enzyme per minute (Chandrasekaran et al, 2015;Majumdar et al, 2015).…”