Recombinant production of Aspergillus Flavus uricase and investigation of its thermal stability in the presence of raffinose and lactose
Aspergillus flavus uricase (Rasburicase) with a molecular mass of 135 kDa is currently used for the treatment of gout and hyperuricemia occurring in tumor lysis syndrome. To characterize the effects of raffinose and lactose osmolytes on the uricase structure, its coding sequence was cloned, expressed in E. coli BL21, and purified by Ni–NTA agarose affinity chromatography. Thermal inactivation studies at 40 °C showed that nearly 15% of UOX activity was preserved, while the presence of raffinose and lactose reduced its activity to 35 and 45% of its original activity, respectively. Investigation of UOX thermal stability at 40 °C in the course of time showed that the enzyme relatively lost almost 60% of its original activity after 40 min, whereas more than 50% of UOX activity is preserved in the presence of lactose. Estimation of thermal inactivation rate constant, k in, showed that the UOX k in and UOX k in in the presence of raffinose was unchanged (0.018 min−1), whereas for the presence of lactose, it was 0.015 min−1. Half-life and T m analysis showed that UOX half-life is almost 38 min and addition of raffinose did not change the half-life, whereas the presence of lactose had remarkable impact on UOX half-life (46 min). The presence of raffinose increased UOX T m to a lesser extent, whereas lactose notably enhanced the T m from 27 to 37 °C. Overall, our findings show that lactose has protective effects on UOX stability, while for raffinose, it is relatively compromised.
KeywordsUricase Osmolyte Thermal stability Recombinant Aspergillus flavus
The authors would like to thank the research council of Urmia University for financial support. Technical support from Mr. Ashkan Basirnia and Mrs. Razieh Pak-Tarmani is highly appreciated, as well.
Compliance with ethical standards
Conflict of interest
The authors report no conflicts of interest.
- Golabi M, Naem S, Imani M, Dalirezh N (2016) Evidence of morphine like substance and μ-opioid receptor expression in Toxacara canis (Nematoda: Ascaridae). In: Veterinary research forum, vol 4. Faculty of Veterinary Medicine, Urmia University, pp 331–335Google Scholar
- Legoux R et al (1992) Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase. J Biol Chem 267:8565–8570Google Scholar
- Mahler H, HuBSCHER G, Baum H (1955) Studies on uricase. J Biol Chem 216:625–641Google Scholar
- Moussa B, Farouk F, Azzazy H (2010) A validated RP-HPLC method for the determination of recombinant human insulin in bulk and pharmaceutical dosage form. J Chem 7:S449–S457Google Scholar
- Pession A, Melchionda F, Castellini C (2008) Pitfalls, prevention, and treatment of hyperuricemia during tumor lysis syndrome in the era of rasburicase (recombinant urate oxidase). Biologics 2:129–141Google Scholar
- Ranga R, Sarada A, Baskaran V, Ravishankar GA (2009) Identification of carotenoids from green alga Haematococcus pluvialis by HPLC and LC-MS (APCI) and their antioxidant properties. J Microbiol Biotechnol 19:1333–1341Google Scholar
- Sharma S, Pathak N, Chattopadhyay K (2012) Osmolyte induced stabilization of protein molecules: a brief review. J Proteins Proteomics 3(2):129–139 Google Scholar