Recombinant production of Aspergillus Flavus uricase and investigation of its thermal stability in the presence of raffinose and lactose

Abstract

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 , showed that the UOX k and UOX k in the presence of raffinose was unchanged (0.018 min), whereas for the presence of lactose, it was 0.015 min. Half-life and T 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 to a lesser extent, whereas lactose notably enhanced the T from 27 to 37 °C. Overall, our findings show that lactose has protective effects on UOX stability, while for raffinose, it is relatively compromised.

Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli

“…The retained activity of uricase from B. subtilis is ~ 50% after incubation at 37 °C for 12 h (Pfrimer et al 2010). Only 15% of uricase activity from A. flavus is preserved after 10-min incubation at 40 °C (Imani and Shahmohamadnejad 2017). In the present study, the specific activity of KmUOX was 50.54 U mg −1 , which is rather high relative to the reported uricases.…”

Molecular Elucidation of a Urate Oxidase from Deinococcus radiodurans for Hyperuricemia and Gout Therapy

“…Some urate oxidases with high specific activity are not so thermostable. The urate oxidase from A. flavus retains only 30% activity after incubation at 40 °C for 1 h; its specific activity is 27 U mg −1 [ 14 , 41 ]. The urate oxidase from C. utilis retains only 40% activity after incubation at 37 °C for 24 h; its specific activity is 38.4 U mg −1 [ 27 ].…”

Enhancement of Thermostability of Aspergillus flavus Urate Oxidase by Immobilization on the Ni-Based Magnetic Metal–Organic Framework

“…It has been suggested that at elevated temperatures (i.e., around and above 40 • C), the tetrameric Uox enzyme tends to disassociate into the inactive monomeric subunits [12]. For example, it was shown that at 35 • C, the Uox could lose its activity to below 20% of its original activity level, and also with any further increase in temperature, the Uox activity may decline further [73]. In addition, this inactivation has also been shown to happen in a concentration-dependent manner.…”