Abstract
The present work reports on the production of extracellular l-asparaginase from Rhizopus microsporus IBBL-2 using submerged fermentation (SmF) process free of glutaminase and urease activities. Primary studies done in shake flask showed that the highest l-asparaginase activity of 12.68 U mL(-1) was produced at 72 h with optimized fermentation parameters such as pH 6.0, 4 × 10(6) fungal cells mL(-1), and agitation of 180 rpm at 30 °C using one-factor-at-a-time (OFAT). Different substrates, nitrogen sources, temperature, pH, the initial number of cells and metal ions were tested to determine the impact on enzyme production. l-Asparaginase activity of 17.68 U mL(-1) was produced after 48 h using immobilized calcium-alginate (Ca-alginate) cells. 4 × 10(6) cells mL(-1) was entrapped in 3% (W/V) of alginate bead of size 2 mm each at a temperature of 30 °C and pH of 6. The process was optimized using L9 (3(4)) Taguchi Orthogonal Array (OA) technique with a regression coefficient (R (2)) value of 0.9709, F value of 33.34 and p value of 0.0025. Scale-up studies involving 200-mL and 1-L rotating bed reactor (RBR) using immobilized beads were done and the results obtained are 20.21 U mL(-1) and 19.13 U mL(-1), respectively, the increased activity with immobilization accounts for reduced shear on cells due to increased stability as compared to the free-flowing cells.