Molecular Biology and Proteomics Laboratory, Department of Biotechnology, Indian Institute of Technology, Roorkee, 247667, India.
Mol Biol Rep. 2020 May;47(5):3251-3270. doi: 10.1007/s11033-020-05374-x. Epub 2020 Apr 15.
Superoxide dismutase is one of the key antioxidant enzymes accountable for the eradication of free radicals generated during various metabolic processes. This is first study reporting a thermostable MnSOD obtained from a xerophytic plant, Nerium oleander. The full-length gene identified using Rapid amplification of cDNA ends revealed an open reading frame of 699 bp flanked by 5'UTR and 3'UTR of 134 bp and 198 bp respectively. The corresponding NeMnSOD protein was cloned and expressed in Escherichia coli. The purified protein yields a band of 25.4 kDa, which established a specific activity of 2617 units mg of protein and under native condition yield bands of 52 kDa and 110 kDa, confirming the dimeric and tetrameric state of the protein. The Km and V of 0.078 ± 0.008 mM and 1052.3 ± 33.59 units mg of protein, respectively. The purified enzyme demonstrated thermostability by retaining more than 20% activity at a temperature 70 ℃. The enzyme functioned at pH range of 4-9.0 with maximum activity at pH 7.4. Sodium azide, effectively inhibited the activity of enzyme confirming it to be MnSOD. The enzyme activity was least affected on treatment with strong denaturants (Urea, guanidine HCl and SDS) and harsh chemicals (DTT, CHAPS and β-mercapto-ethanol) These experimental data validated with Insilco analysis revealed that NeMnSOD possessed thermo as well as kinetically stable moiety which can be further exploited with its applications in the field of pharmaceutical, food and cosmetic industry, which urge for such thermostable enzyme.
超氧化物歧化酶是负责消除各种代谢过程中产生的自由基的关键抗氧化酶之一。这是首次报道从旱生植物夹竹桃中获得的耐热 MnSOD。使用快速 cDNA 末端扩增鉴定全长基因，发现一个 699bp 的开放阅读框，其侧翼分别为 5'UTR 和 3'UTR，长度分别为 134bp 和 198bp。克隆并在大肠杆菌中表达相应的 NeMnSOD 蛋白。纯化的蛋白产生 25.4kDa 的条带，其比活为 2617 单位 mg 的蛋白，在天然条件下产生 52kDa 和 110kDa 的条带，证实了蛋白的二聚体和四聚体状态。Km 和 V 分别为 0.078±0.008mM 和 1052.3±33.59 单位 mg 的蛋白。纯化的酶表现出热稳定性，在 70℃时保留超过 20%的活性。该酶在 pH 值为 4-9.0 的范围内发挥作用，在 pH 值为 7.4 时具有最大活性。叠氮化钠有效抑制酶的活性，证实其为 MnSOD。在处理强变性剂（尿素、盐酸胍和 SDS）和苛刻化学物质（DTT、CHAPS 和 β-巯基乙醇）时，酶活性受影响最小。这些实验数据与 Insilco 分析结果一致，表明 NeMnSOD 具有热稳定性和动力学稳定性的部分，可进一步应用于制药、食品和化妆品行业，这些行业对这种耐热酶的需求迫切。
