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Exploring the In Vitro Thrombolytic Activity of Nattokinase From a New Strain Pseudomonas aeruginosa CMSS.

Chandrasekaran SD, Vaithilingam M, Shanker R, Kumar S, Thiyur S, Babu V, Selvakumar JN, Prakash S - Jundishapur J Microbiol (2015)

Bottom Line: 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.To enhance the yield of NK, effect of various parameters such as pH, temperature, carbon source, nitrogen source and inoculum size were optimized.This study is unique and the findings are the first report on the production of NK from P. aeruginosa CMSS isolated from cow milk.

View Article: PubMed Central - PubMed

Affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.

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.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic Tree of Pseudomonas aeruginosa CMSS
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fig23413: Phylogenetic Tree of Pseudomonas aeruginosa CMSS

Mentions: The CMSS isolate was found to be a motile and gram-negative rod. The biochemical characteristics of the isolate were as follows; catalase (+), Oxidase (+), Indole (-), Methyl red (-), Voges-Prausker (VP) (-), citrate utilization (+), urease (+), nitrate reduction (+), glucose (-), sucrose (-), lactose (-) and maltose (+). Based on the morphological and biochemical characterization, the CMSS strain was identified as Pseudomonas sp. The 16S rRNA sequencing was exported to the database and checked for homologous alignment. Based on the alignment results, the CMSS strain was confirmed as P. aeruginosa, which showed 99% similarity with other Pseudomonas sp. The partial 16S rRNA sequences were deposited in Gen Bank under the accession number JX112657. Aphylogenetic tree was constructed for the CMSS strain with bootstrap values (Figure 1). The phylogenetic tree based on 16S rRNA sequences showed that the isolate occupies a distinct phylogenetic position within the P. aeruginosa family. Based on the molecular taxonomy and phylogeny, the strain was identified as P. aeruginosa and designated as P. aeruginosa CMSS.


Exploring the In Vitro Thrombolytic Activity of Nattokinase From a New Strain Pseudomonas aeruginosa CMSS.

Chandrasekaran SD, Vaithilingam M, Shanker R, Kumar S, Thiyur S, Babu V, Selvakumar JN, Prakash S - Jundishapur J Microbiol (2015)

Phylogenetic Tree of Pseudomonas aeruginosa CMSS
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644271&req=5

fig23413: Phylogenetic Tree of Pseudomonas aeruginosa CMSS
Mentions: The CMSS isolate was found to be a motile and gram-negative rod. The biochemical characteristics of the isolate were as follows; catalase (+), Oxidase (+), Indole (-), Methyl red (-), Voges-Prausker (VP) (-), citrate utilization (+), urease (+), nitrate reduction (+), glucose (-), sucrose (-), lactose (-) and maltose (+). Based on the morphological and biochemical characterization, the CMSS strain was identified as Pseudomonas sp. The 16S rRNA sequencing was exported to the database and checked for homologous alignment. Based on the alignment results, the CMSS strain was confirmed as P. aeruginosa, which showed 99% similarity with other Pseudomonas sp. The partial 16S rRNA sequences were deposited in Gen Bank under the accession number JX112657. Aphylogenetic tree was constructed for the CMSS strain with bootstrap values (Figure 1). The phylogenetic tree based on 16S rRNA sequences showed that the isolate occupies a distinct phylogenetic position within the P. aeruginosa family. Based on the molecular taxonomy and phylogeny, the strain was identified as P. aeruginosa and designated as P. aeruginosa CMSS.

Bottom Line: 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.To enhance the yield of NK, effect of various parameters such as pH, temperature, carbon source, nitrogen source and inoculum size were optimized.This study is unique and the findings are the first report on the production of NK from P. aeruginosa CMSS isolated from cow milk.

View Article: PubMed Central - PubMed

Affiliation: Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore, India.

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.

No MeSH data available.


Related in: MedlinePlus