Department of Biotechnology, Institute of Science, High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman-Iran, Iran.
Department of chemistry, Faculty of science, University of Birjand, Birjand, Iran.
J Biomol Struct Dyn. 2022 Mar;40(4):1461-1471. doi: 10.1080/07391102.2020.1828167. Epub 2020 Oct 1.
Urate oxidase (EC 1.7.3.3) is a key enzyme in the purine metabolism which is applied in the treatment of gout and also, as a diagnostic reagent for uric acid detection. In the current study, the trehalose (TRE) effects as an additive on the structural stability and function of uricase were investigated. For recombinant expression of UOX in E. coli BL21 cells, firstly the coding sequence was subcloned into the pET-28a vector and after induction with IPTG, the recombinant UOX was purified by affinity chromatography using a Ni-NTA agarose column. To specify the trehalose effects on the urate oxidase (UOX) structure, optimum pH, optimum temperature, kinetic and thermodynamic parameters and also, the intrinsic fluorescence of UOX in the absence and presence of trehalose were examined. The UOX half-life is 24.32 min at 40 °C, whereas the UOX-TRE has a higher half-life (32.09 min) at this temperature. Generally, our findings confirm that trehalose has a protective effect on the enzyme structure. Optimum pH and temperature were 9 and 25 °C, respectively for both the naked and treated enzymes and their activity retained 42.18 and 64.80%, respectively after 48 h of incubation at room temperature. Also, theoretical results indicate that the random coil of the enzyme was converted to α-helix and β-sheet in the presence of trehalose which may preserve the integrity of the active site of the enzyme and increased the enzymatic activity. The MD simulation results indicated greater stability of the uricase structure in the presence of trehalose.Communicated by Ramaswamy H. Sarma.
尿酸氧化酶（EC 1.7.3.3）是嘌呤代谢中的关键酶，可用于治疗痛风，也可用作尿酸检测的诊断试剂。在本研究中，研究了海藻糖（TRE）作为添加剂对尿酸酶结构稳定性和功能的影响。为了在大肠杆菌 BL21 细胞中重组表达 UOX，首先将编码序列亚克隆到 pET-28a 载体中，然后用 IPTG 诱导，通过 Ni-NTA 琼脂糖柱亲和层析纯化重组 UOX。为了确定海藻糖对尿酸氧化酶（UOX）结构的影响，研究了最适 pH 值、最适温度、动力学和热力学参数以及在不存在和存在海藻糖的情况下 UOX 的本征荧光。UOX 在 40°C 下的半衰期为 24.32 分钟，而 UOX-TRE 在该温度下具有更高的半衰期（32.09 分钟）。一般来说，我们的研究结果证实了海藻糖对酶结构具有保护作用。最适 pH 值和温度分别为 9 和 25°C，对于裸酶和处理酶都是如此，在室温下孵育 48 小时后，其活性分别保留了 42.18%和 64.80%。此外，理论结果表明，在海藻糖存在下，酶的无规卷曲转变为α-螺旋和β-折叠，这可能保持酶活性部位的完整性并提高酶活性。MD 模拟结果表明，在海藻糖存在下尿酸酶结构更稳定。由 Ramaswamy H. Sarma 交流。
