来自嗜热栖热放线菌亚种ATCC 393的重组L-天冬酰胺酶II及其抗癌活性。
Recombinant l-Asparaginase II from subsp. ATCC 393 and Its Anticancer Activity.
作者信息
Aishwarya S Susan, Selvarajan E, Iyappan S, Rajnish K N
机构信息
Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Tamilnadu India.
出版信息
Indian J Microbiol. 2019 Sep;59(3):313-320. doi: 10.1007/s12088-019-00806-0. Epub 2019 Apr 23.
l-asparaginases from bacterial origin are employed extensively in leukemic treatment and food industry. The present study focuses on the characterization of the recombinant l-asparaginase II from subsp. ATCC 393 cloned into expression system and purified using Ni-NTA chromatography. The recombinant l-asparaginase as a monomer had a molecular weight of 35 kDa. The enzyme was active from 10 to 80 °C with the optimum at 40 °C. The enzyme retained its activity at 28 °C and 37 °C up to 24 h. The enzyme had optimum pH of 6 and retained 50% activity till 18 h. The K of the recombinant enzyme was 0.01235 mM and V 1.576 mM/min. The half life of recombinant l-asparaginase II in human serum was 44 h and trypsin was for 15 min. The LC-MS/MS analysis revealed the molecular weight of 35,050 and pI of 5.64. The secondary structure prediction using CD spectroscopy for the recombinant enzyme showed 33.5% α-helix, 66.5% turn and 0% β sheets. The cytotoxicity of the recombinant enzyme was analysed against MOLT 3, Jurkat E6.1 and K-562 with the IC 50 value of 30, 62.5 and 50 µg/ml.
源自细菌的L-天冬酰胺酶广泛应用于白血病治疗和食品工业。本研究聚焦于对克隆到表达系统中并通过镍-氮三乙酸（Ni-NTA）色谱法纯化的来自亚种ATCC 393的重组L-天冬酰胺酶II进行特性分析。重组L-天冬酰胺酶作为单体，分子量为35 kDa。该酶在10至80°C具有活性，最适温度为40°C。该酶在28°C和37°C下活性可保持24小时。该酶的最适pH为6，活性保持50%直至18小时。重组酶的K m为0.01235 mM，V max为1.576 mM/分钟。重组L-天冬酰胺酶II在人血清中的半衰期为44小时，在胰蛋白酶中为15分钟。液相色谱-串联质谱（LC-MS/MS）分析显示分子量为35,050，等电点为5.64。使用圆二色光谱（CD）对重组酶进行二级结构预测显示，α-螺旋占33.5%，转角占66.5%，β-折叠占0%。分析了重组酶对MOLT 3、Jurkat E6.1和K-562的细胞毒性，IC 50值分别为30、62.5和50 μg/ml。
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