Extracellular urease from Arthrobacter creatinolyticus MTCC 5604: scale up, purification and its cytotoxic effect thereof

Abstract

Urease is a potent metalloenzyme with diverse applications. This paper describes the scale up and purification of an extracellular urease from Arthrobacter creatinolyticus MTCC 5604. The urease production was scaled-up in 3.7L and 20L fermentor. A maximum activity of 27 and 27.8U/mL and a productivity of 0.90 and 0.99 U/mL/h were obtained at 30h and 28h in 3.7 and 20L fermentor, respectively. Urease was purified to homogeneity with 49.85-fold purification by gel filtration and anion exchange chromatography with a yield of 36% and a specific activity of 1044.37 U/mg protein. The enzyme showed three protein bands with molecular mass of 72.6, 11.2 and 6.1kDa on SDS-PAGE and ~ 270kDa on native PAGE. The cytotoxic effect of urease was assessed in vitro using cancer cell lines (A549 and MG-63) and normal cell line (HEK 293). Urease showed its inhibitory effects on cancer cell lines through the generation of toxic ammonia, which in turn increased the pH of the surrounding medium. This increase in extracellular pH, enhanced the cytotoxic effect of weak base chemotherapeutic drugs, doxorubicin (50µM) and vinblastine (100µM) in the presence of urease (5U/mL) and urea (0-4mM) significantly.