Separation of L-asparaginase from halophilic Bacillus subtilis bacteria native to Iran

Abstract

L-Asparaginase (L-asparagine amid hydrolase; EC 3.5.1.1) catalyzes the deamination of l-asparagine into l-aspartate and ammonia. L-Asparaginase exists in various organisms, including animals, plants, yeast, fungi, bacteria, and archaea and humans. It can be used as an important chemotherapeutic agent for the treatment of a variety of disorders such as acute lymphoblastic leukemia, malignancy of the lymphoid system, and Hodgkin’s lymphomas. Also, another important application is in the food industry, by using the properties of this enzyme to reduce acrylamide levels in commercially fried foods, maintaining their characteristics (color, flavor, texture, security, etc.).Aim: The purpose of this study is the separation of L-Asparaginase from Halophilic Bacillus Subtilis bacteria native to Iran (bacillus Subtilis D6A and DAR). Method: We used the PEG precipitation method with various percentages and molecular weights of PEG. The bacteria samples were cultured in a broth nutrient medium. After incubation (37 ,5 days) of microorganisms, the culture medium containing the enzyme was separated from the cells by centrifugation at 5000 rpm for 10 minutes at room temperature. The total protein content is measured by the Bradford method. In the next step, using the polyethylene glycol with different molecular weights (4,6,8,10 KDa) at various percentages and at pH:6.5, the l asparaginase was precipitated. SDS-PAGE is applied to analyse the purity of precipitated samples. Result:The following conditions had better results in percentages l'asparaginase produced by the D6A and DAR respectively: by PEG 4000 and 6000,10-20 percentages, and by PEG 8000 and 10000,5-10 and 10-15 percentages.Discussion: Precipitation by PEG can be considered as an easy, cost-effective and suitable method to start purification processes and downstream steps of L-Asparaginase extraction produced by bacteria.