Validation of a Simple and Rapid Method for Assessment of Intracellular Bacterial Asparaginase

Document Type: Research Paper

Authors

1 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Pharmaceutical Sciences research center, shahid beheshti university of medical sciences, Tehran, iran

4 Department of Medical Biotechnology, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran

5 Department of Pharmaceutical Biotechnology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Abstract

L-Asparaginase has remarkable properties which make it useful in dual pharmaceutical and food industries.In this study, simple and advantageous method has been validated for rapid and precise determination of intracellular L-Asparaginasein bacterial species. A suspension of bacterial cells was used instead ofcell extract and incubated by substrate (asparagine) after simple wash and centrifugation steps. Due to loss of enzyme activity which can cause by cell distruption methods such as sonication or enzymatic treatment, cell suspension were used instead of the cell extrac. Thus, this method not onlyis cost effective but also speed up the screening process and lead to higher measurement accuracy. To validate this method, two species of bacteria E.coli ATCC 8739 and Halomonas H28 were used. After cultivation, the cells were harvested and washed. Then, 5 serial dilutions were prepared from each bacterium, and the asparaginase activity in each of them measured by methods includes, sonication, enzymatic lyses and the cell suspension. The results have showed that the changes in asparaginase activity in all 5 serial dilutions are linear and there is good agreement between the sonication and the cell suspension methods. Also, it was shown that activities measured by the enzymatic method were significantly higher than the othertwo methods.

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