Isolation, Identification, and Optimization of L-asparaginase Production from Enterobacter hormaechei through Submerged Fermentation Using Response Surface Methodology

BACKGROUND AND OBJECTIVESL-asparaginase, an enzyme responsible for converting L-asparagine into L-aspartic acid and ammonia has been employed as a chemotherapeutic agent for treating acute lymphoblastic leukemia. This enzyme's clinical effectiveness stems from its ability to reduction L-asparagine levels. Due to its therapeutic benefits, the discovery of microbial sources producing asparaginase has seen a significant increase in recent years. The objective of this study was to isolate and identify bacteria capable of producing the L-asparaginase from different samples and optimization the influential parameters affecting on its production.MATERIALS AND METHODSThe isolation of bacteria producing asparaginase from diverse samples such as soil, water, and dairy products was conducted using a specialized M۹ medium enriched with phenol red and asparagine. Additionally, to assess the glutaminase activity in bacteria that exhibited a positive asparaginase test, a medium containing phenol red and glutamine was employed. The identification of bacterial isolates with positive asparaginase and negative glutaminase activity was achieved through a combination of morphological, biochemical, and ۱۶S rRNA methods. Enzyme activity was measured using a colorimetric method. Then the factors affecting enzyme production such as carbon and nitrogen sources, temperature, and pH were optimized by Response Surface Method.RESULTS AND DISCUSSIONOut of the ۱۹ enzyme-producing isolates screened on M۹ agar, four were identified as excellent producers. Among these, only one isolate exhibited a lack of glutaminase activity. Analysis of the ۱۶S rRNA sequences of these isolates revealed a remarkable ۹۹.۶% similarity with Enterobacter hormaechei. Finest enzyme activity was ۶.۷۸ IU/ml. Further, the L-asparaginase production was optimized using Response Surface Methodology (RSM). This approach led to the highest observed asparaginase activity, reaching ۱۷.۵۱ IU/ml. This optimal activity was achieved by utilizing glucose as the carbon source, ammonium chloride as the nitrogen source, temperature of ۳۷°C, and the pH at ۷.CONCLUSIONThe study explored the Enterobacter hormaechei as a potent and potential bacterial source for high yield of anti-leukemic drug.

کلیدواژه ها:

L-asparaginase ، Enterobacter hormaechei ، Anti-leukemic drug ، Optimization