Vol.:(0123456789) 1 3 Molecular Biology Reports https://doi.org/10.1007/s11033-018-4453-8 ORIGINAL ARTICLE Extracellular urease from Arthrobacter creatinolyticus MTCC 5604: scale up, purifcation and its cytotoxic efect thereof Ramesh Rajendran 1  · Ajitha Pandi 1  · Aparna Ramchary 1  · Hemalatha Thiagarajan 1  · Jithendra Panneerselvam 2  · Ayyadurai Niraikulam 1  · Gowthaman Marichetti Kuppuswami 1  · Kamini Numbi Ramudu 1 Received: 5 September 2018 / Accepted: 18 October 2018 © Springer Nature B.V. 2018 Abstract Urease is a potent metalloenzyme with diverse applications. This paper describes the scale up and purifcation of an extracel- lular urease from Arthrobacter creatinolyticus MTCC 5604. The urease production was scaled-up in 3.7 L and 20 L fermentor. A maximum activity of 27 and 27.8 U/mL and a productivity of 0.90 and 0.99 U/mL/h were obtained at 30 h and 28 h in 3.7 and 20 L fermentor, respectively. Urease was purifed to homogeneity with 49.85-fold purifcation by gel fltration and anion exchange chromatography with a yield of 36% and a specifc activity of 1044.37 U/mg protein. The enzyme showed three protein bands with molecular mass of 72.6, 11.2 and 6.1 kDa on SDS-PAGE and ~ 270 kDa on native PAGE. The cytotoxic efect of urease was assessed in vitro using cancer cell lines (A549 and MG-63) and normal cell line (HEK 293). Urease showed its inhibitory efects 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 efect of weak base chemotherapeutic drugs, doxorubicin (50 µM) and vinblastine (100 µM) in the presence of urease (5 U/mL) and urea (0–4 mM) signifcantly. Keywords Arthrobacter creatinolyticus · Chemotherapeutic drugs · Cytotoxicity · Purifcation · Scale-up · Urease Introduction Urease (urea amidohydrolase; EC 3.5.1.5), a nickel— dependent metalloenzyme catalyses the hydrolysis of urea into ammonia and carbon dioxide. Urease has many indus- trial applications, such as diagnostic kits for measuring urea [1], in alcoholic beverages [2], in biosensors of haemodialy- sis systems for determining blood urea [3, 4] and as a probe for detection of heavy metal ions in food sample, ground water, industrial efuents and environmental monitoring [5, 6]. Ureases are multifunctional toxic proteins which are also gaining importance in the feld of crop protection, animal and human diseases [7]. Urease has been recognized as an important virulence factor in the pathological studies and the ability of the enzyme to hydrolyze urea could be used as a marker to predict its cytotoxic efects on various cell lines. The cytotoxic efect of urease was reported with ureases from Vibrio parahaemolyticus [8], Helicobacter pylori [9] and jack bean urease [10] on various cell lines including, human erythro leukaemia cell line K562, A549 and MDA- MB-231 cell lines, respectively. In general, solid tumors produce a local microenvironment which is more acidic than normal tissues [11, 12]. The low pH in tumors could reduce the efectiveness of small molecule chemothera- peutics, thereby decreasing its cytotoxicity. The efcacy of weak-base drugs such as mitoxantrone, daunorubicin and doxorubicin could be enhanced by raising the extracellular pH [13]. This increased alkalinization may counteract the adverse efect of the acidic microenvironment on weakly basic drugs and enhances the uptake of these chemothera- peutics [10]. Ureases are wide-spread in plants, fungi and bacteria. Microbial ureases are intracellular in nature and are prepared either by sonication of the cells [14, 15] or by homogeniza- tion of the mycelia [16, 17]. Ramesh et al. [18] reported the production of an extracellular urease from Arthrobacter creatinolyticus MTCC 5604. The partially purifed urease * Kamini Numbi Ramudu nrkamini@redifmail.com 1 Department of Biochemistry and Biotechnology, CSIR - Central Leather Research Institute, Adyar, Chennai 600 020, India 2 Department of Pharmaceutical Technology, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia