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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

Effect of (a) Carbon Source (b) Nitrogen Source and (c) Inoculum Size on Nattokinase Production
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fig23415: Effect of (a) Carbon Source (b) Nitrogen Source and (c) Inoculum Size on Nattokinase Production

Mentions: In our preliminary experiments, the potent isolate Pseudomonas sp. CMSS in the production medium showed maximum enzyme productivity with maximum protein content (2.7 mg/mL) and enzyme activity (1045 U/mL). Different parameters, pH and temperature affect various biological processes and play important roles in the optimization process. The optimum enzyme activity was observed at pH 7 (1514 U mL-1 and 2.8 mg mL-1) and 25˚C (1532 U mL-1 and 2.8 mg mL-1). However, after a certain optimum percentage, the addition of further inoculum negatively affects fermentation by decreasing the dilution rate (Figure 2). Based on the obtained results, the optimum carbon source for the enzyme productivity of the P. aeruginosa CMSS strain was found to be 1% sucrose, in the presence of which maximum NK activity of 1721 U mL-1 was observed. Much less enzyme activity was obtained in the medium supplemented with glycerol (518 U mL-1). Furthermore, P. aeruginosa CMSS grown on shrimp shell powder supplemented medium, showed better activity (2524 U mL-1) than the strain grown in medium with other nitrogen supplements. The inoculum concentration of 1% was more favorable than the generally employed concentrations of up to 3%. The protein content and enzyme activity was found extremely high with the inoculum amount of 1% (2581 U mL-1). The production rate gradually increased with the increase in inoculum percentage (Figure 3).


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)

Effect of (a) Carbon Source (b) Nitrogen Source and (c) Inoculum Size on Nattokinase Production
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig23415: Effect of (a) Carbon Source (b) Nitrogen Source and (c) Inoculum Size on Nattokinase Production
Mentions: In our preliminary experiments, the potent isolate Pseudomonas sp. CMSS in the production medium showed maximum enzyme productivity with maximum protein content (2.7 mg/mL) and enzyme activity (1045 U/mL). Different parameters, pH and temperature affect various biological processes and play important roles in the optimization process. The optimum enzyme activity was observed at pH 7 (1514 U mL-1 and 2.8 mg mL-1) and 25˚C (1532 U mL-1 and 2.8 mg mL-1). However, after a certain optimum percentage, the addition of further inoculum negatively affects fermentation by decreasing the dilution rate (Figure 2). Based on the obtained results, the optimum carbon source for the enzyme productivity of the P. aeruginosa CMSS strain was found to be 1% sucrose, in the presence of which maximum NK activity of 1721 U mL-1 was observed. Much less enzyme activity was obtained in the medium supplemented with glycerol (518 U mL-1). Furthermore, P. aeruginosa CMSS grown on shrimp shell powder supplemented medium, showed better activity (2524 U mL-1) than the strain grown in medium with other nitrogen supplements. The inoculum concentration of 1% was more favorable than the generally employed concentrations of up to 3%. The protein content and enzyme activity was found extremely high with the inoculum amount of 1% (2581 U mL-1). The production rate gradually increased with the increase in inoculum percentage (Figure 3).

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