Production and purification of Nattokinase from Pseudomonas aeruginosa P49

Shahad Abdullah Shwan, Ali Jabbar Reshak

Abstract


Background: Nattokinase (NK) is a profibrinolytic serine protease enzyme produced by many bacterial strains, such as Pseudomonas aeruginosa. Therefore, this study aimed to produce NK from local isolate of P. aeruginosa P49 and optimize its conditions for the production of enzyme.

Methods: 150 samples were obtained from clinical sources, during the period from August to November 2022, from different hospitals. All samples were subjected to different examinations, in addition to VITEK2 system, to confirm that these isolates were P. aeruginosa.

Result: A total of one hundred P. aeruginosa isolates were screened to choose the best NK-producing isolates using skim milk agar, then broth media, whereas P. aeruginosa P49 gave the highest enzymatic activity (337.9 U/mg protein). Optimal conditions for the formation of NK were estimated, and the results showed that the maximum production of NK was gained using peptone-yeast medium containing sucrose, peptone, and CaSO4.2H2O at pH 7.5 and 37°C for 24 hours of incubation, whereas the activity of NK increased to reach a yield of 1603 U/mg protein. The NK was purified from P. aeruginosa P49 utilizing ion exchange chromatography (IEC) after precipitation by ammonium sulfate (0-75%). The results for enzyme purification gave 96% of NK enzyme with a purification fold of 3.6, and the specific activity was 2190.7 U/mg protein.

Conclusion: This result suggests P. aeruginosa P49 is a good source of NK production.

Keywords: Nattokinase; Pseudomonas aeruginosa; Enzyme Purification; IEC; SF; VITEK2 system; Ion exchange Chromatography


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References


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DOI: http://dx.doi.org/10.62940/als.v12i4.2760

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