Imani Mehdi, Shahmohamadnejad Serveh
Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran.
3 Biotech. 2017 Jul;7(3):201. doi: 10.1007/s13205-017-0841-3. Epub 2017 Jun 30.
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.
分子量为135 kDa的黄曲霉尿酸酶（拉布立酶）目前用于治疗肿瘤溶解综合征中出现的痛风和高尿酸血症。为了表征棉子糖和乳糖渗透剂对尿酸酶结构的影响，克隆了其编码序列，在大肠杆菌BL21中表达，并通过镍-亚氨基二乙酸琼脂糖亲和色谱法进行纯化。40℃下的热失活研究表明，近15%的尿酸氧化酶（UOX）活性得以保留，而棉子糖和乳糖的存在分别将其活性降低至原始活性的35%和45%。在40℃下随时间对UOX热稳定性的研究表明，该酶在40分钟后相对丧失了近60%的原始活性，而在乳糖存在下，超过50%的UOX活性得以保留。热失活速率常数k的估计表明，在棉子糖存在下UOX的k不变（0.018 min⁻¹），而在乳糖存在下，其为0.015 min⁻¹。半衰期和T₁/₂分析表明，UOX半衰期约为38分钟，添加棉子糖不会改变半衰期，而乳糖的存在对UOX半衰期有显著影响（46分钟）。棉子糖的存在在较小程度上提高了UOX的T₁/₂，而乳糖则显著将T₁/₂从27℃提高到37℃。总体而言，我们的研究结果表明，乳糖对UOX稳定性具有保护作用，而棉子糖的保护作用相对较弱。
