Bacillus subtilis as an expression host for the production of glutaminase free recombinant L-asparaginase II

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The bacterial L-asparaginase has been widely used as a therapeutic agent in the treatment of ALL (acute lymphoblastic leukemia). Moreover, it is used in food industry for the production of acrylamide free starchy/baked foods, L-asparagine biosensor for diagnosis of leukemia and as a model enzyme for the development of new drug delivery systems. The various side effects of L-asparaginases are mainly due to the presence of partial glutaminase activity. Hence, we made an attempt to produce glutaminase-free L-asparaginase II, which is highly desirable for its successful application.The gene encoding glutaminase-free L-asparaginase II (ans B2) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43 vector and transformed in Bacillus subtilis WB800N. It was further optimized to maximize the expression levels of recombinant enzyme (rL-asp II). A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that > 90 % of recombinant enzyme is secreted extracellularly. The expression of recombinant L-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilized metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that an agitation (120 rpm), temperature (37 ºC), Isopropyl b-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (at 0.8 OD600nm) plays a vital role in achieving a maximum of 55 IU/ml. Furthermore, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml of protein.
Supervisor: Veeranki Venkata Dasu