Efficient production of L-asparaginase using recombinant Pichia pastoris and strategy to eliminate glutaminase activity.

The mortality rate of Acute Lymphoblastic Leukemia (ALL) has reduced after the incorporation of L-asparaginase (L-ASNase) in therapeutic regimes, which function via selective starvation of the cancer cells, sparing the rest. However, challenges persist due to the immunogenic nature of the enzyme and the co-glutaminase activity. This study investigates the L-ASNase coded for by the ansB gene from Serratia marcescens MTCC 97. An initial study led to a titer of 1.6 U/mL of L-ASNase in a semi synthetic media. However, due to the opportunistic pathogenic nature of the strain and lower yield of the enzyme, a robust eukaryotic host, and the Pichia pastoris GlycoSwitch® SuperMan5 (pep4-prb1-) strain was selected for the expression. The gene was codon optimized to further enhance the yield. The recombinant strain produced 16.6 U/mL of L-ASNase in a complex BMMY medium. Process and parameter optimization were done via statistical design using the Taguchi orthogonal array, leading to a final yield of 22.3 U/mL of L-ASNase, obtained at 37 °C, at a strictly neutral pH, when 1% inoculum was used to initiate the fermentation, with 0.5% v/v methanol induction. Batch bioreactor cultivation further enhanced the L-ASNase titer to 31.75 U/mL. However, the L-ASNase was also found to have a glutaminase activity, for which in-silico characterization of the enzyme was done for an elevated understanding of the intricate structure and active sites. Through modelling and simulation studies, possible mutations were explored with an aim to reduce the glutaminase activity, and thereby reduce the secondary function of the L-ASNase.