Optimization Studies on Nattokinase enzyme Production by a Mutant Strain Pseudomonas aeruginosa CMSS Isolated From Bovine Milk
1Assistant Professor (Senior), Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore- 632 014, Tamil Nadu, India
2School of Biosciences and Technology, Vellore Institute of Technology University, Vellore, Tamil Nadu, India.
*Corresponding Author E-mail: csubathradevi@vit.ac.in, subaresearch@rediffmail.com
ABSTRACT:
Nattokinase is an enzyme with strong fibrinolytic activity to prevent thrombolytic diseases. Nattokinase is a serine protease; it founds much application in medical industry widely. Nattokinase was purified from the culture supernatant of mutant strain Pseudomonas aeuroginosa CMSS. The optimum pH and temperature for nattokinase production was found to be 6 and 25oC respectively. Production medium supplemented with 1 % sucrose and 1 % SSP showed maximum nattokinase yield and activity.
KEYWORDS: Nattokinase, Pseudomonas aeruginosa CMSS, clot buster, optimization.
INTRODUCTION:
Fibrinolytic drugs have been extensively used in the therapeutic treatment of thrombosis. The major thrombolytic agents are plasminogen activators, such as tPA (tissue-type plasminogen activator), urokinase and streptokinase. WHO1 reported circulatory disorders has led to higher mortality and fatality all over the globe among other communicable and non-communicable diseases. Cardiovascular causes 15 millions of death each year2. In India, 31.7% of deaths occur due to myocardial infarction. Incidence of cardiovascular disease was about 7% in 1970 and increased up to 32% in 2011 in India3.The failure of fibrin degradation by the vascular system leads to thrombus in blood vessels4. Many commercial drugs that paved way to cleave fibrin and also increases side-effects. Most of the bacteria, actinomycetes, algae, and fungi have been found to have fibrin-digestion abilities. Nattokinase (NK) has higher potency to lyse blood clots among other plasminogen activators. NK is primarily isolated from a traditional fermented soybean food as “Natto” in Japan5. It is a subtilisin like serine protease with molecular weight of 27-42 KDa6- 9. It degrades fibrin as well induces tissue plasminogen activator (tPA) and urokinase to activate plasminogen into plasmin and inactivates a plasminogen activator inhibitor10-13.
The present study focus on production of nattokinase from UV mutated P. aeruginosa CMSS. The study updates the enhanced production of NK by media optimization studies using physical and biological parameters by classical method.
MATERIALS AND METHOD:
Production of Nattokinase
Mutant strain
Pseudomonas aeruginosa CMSS (Gen Bank accession number: JX112657) isolated from bovine milk was used in this study. The production medium of 100mL supplemented with shrimp shell powder14was prepared in 250 mL Erlemeyer flask .The medium was inoculated with 1 % inoculum size of P.aeruginosa CMSS and incubated in orbital shaker for 48h at 37˚C at pH 7. After incubation the medium was centrifuged (4˚C, 10,000 rpm for 20 min) and the supernatant was purified by chromatography.
Protein determination
Protein content was determined by Lowry’s method using standard Folin Ciocalteau15.
Enzyme assay
Nattokinase activity was determined by casein digestion method. The enzyme activity was determined using standard graph of tyrosine. One unit of caseinolytic activity16 (U) was defined as the amount of enzyme releasing 1 µM of tyrosine equivalent min-1.
Optimization of pH and temperature
The production medium was optimized at different pH and temperature for enhanced enzyme activity. The pH of the medium was adjusted in the range of 5, 6, 7, 8 and 9 as well as temperature was optimized at 10oC, 20oC, 37oC and 45oC. The culture supernatant was collected after centrifugation and measured for enzyme activity and protein.
Optimization of carbon and nitrogen source
Four kinds of carbon and nitrogen sources were estimated with one-at-a time strategy. Carbon sources were glucose, sucrose, maltose and glycerol and nitrogen sources were shrimp shell powder, yeast extract, casein, and tryptose all of which had equivalent (1%) nitrogen source for carbon determination and vice-versa (according to Wang et al17) for the production medium. The medium was centrifuged and determined for protein content and enzyme activity.
RESULTS AND DISCUSSION:
Mutant P. aeruginosa CMSS was isolated from bovine milk using shrimp shell powder18 as the sole carbon/nitrogen sources. The activity in basal medium containing additional carbon/nitrogen sources of 1% (w/v) SSP was more suitable for nattokinase production. The production media was found to serve as an excellent growth medium for Pseudomonas aeruginosa CMSS strain which efficiently induce the production of fibrinolytic enzyme nattokinase. The fermentation in the standard medium resulted in good amount of microbial biomass production and ultimately higher yield of enzyme was obtained. (Fig 1)
The effect of pH and temperature plays a major role in the production of nattokinase. The enzyme produced with varied activity in different pH and temperature from 5 -9 and 10-45oC respectively. The maximum nattokinase activity (728.8 U mL-1) and the protein content (3.0 mg mL-1) was found at pH 6.0. (Fig 1 a).The maximum activity (750 U mL-1) and protein content (3.2 mgmL-1) was found at 25 °C. (Fig 1 b)
The vital components utilized by the micro-organisms in the surrounding medium plays an important role to enhance the production of enzyme. Among the selected carbon sources, the medium with 1% sucrose expressed maximum nattokinase activity (983 U mL-1) and the protein content (2.3 mg mL-1) (Fig 1 c). The nitrogen supplement 1% SSP (shrimp shell powder) in the medium yielded maximum production of nattokinase (1119 UmL-1) and the protein content (1.8 mgmL-1) (Fig 1 d). Previous results showed that, Bacillus sp. KA38 and Pseudomonas sp. TKU015 expressed maximum nattokinase activity at pH719,20.The medium supplemented with peptone, CaCl2 and yeast extract resulted in higher yield of nattokinase activity exhibited from Bacillus sp.21. B. subtilis produced maximum nattokinase in the medium supplemented with glucose, peptone, CaCl2 and MgCl222. Optimization of nattokinase from Bacillus subtilis (NCIM 2724) reported by Rasangya and Vangalpatti23, as incubation time-24h, inoculum size-1% and pH-7.5 and production of maximum nattokinase was found to be 0.31 mgmL-1. Nattokinase has been produced from different strains of bacteria, fungi and actinomycetes such as Bacillus subtilis IMR-NK1, Bacillus natto NLSSE, Bacillus amyloliquefaciens DC-4, B. subtilis QK02, B. subtilis BK-17, B. subtilis LD-8547, Streptomyces megasporus SD5, Rhizopus chinensis 12, Cordyceps militaris and Fusarium sp. BLB. However, there are very few literatures have been reported about nattokinase produced from Pseudomonas sp. isolated from soil. But the current study reports the production and the optimization of nattokinase from a milk isolate Pseudomonas aeruginosa CMSS.
Fig 1. Effect of process parameters on nattokinase production(a) pH (b) temperature (c) carbon source (d) nitrogen source
CONCLUSION:
Results of the current study proved that UV mutated strain P. aeruginosa CMSS showed maximum nattokinase activity. Large scale industrial production of nattokinase from CMSS has been exploited. The optimization of temperature, pH, carbon and nitrogen sources can reduce the cost of NK production in a bioprocess manufacturing system. In future, in-vivo studies in nattokinase, cloning, sequencing and expression can be focused to optimize better solution for the treatment of cardiovascular diseases.
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Received on 10.09.2014 Modified on 15.09.2014
Accepted on 19.09.2014 © RJPT All right reserved
Research J. Pharm. and Tech. 7(11): Nov. 2014 Page 1297-1299