Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.
Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
Appl Microbiol Biotechnol. 2017 Oct;101(19):7227-7238. doi: 10.1007/s00253-017-8456-5. Epub 2017 Aug 11.
L-asparaginase has been used in the treatment of patients with acute lymphoblastic leukemia (ALL) for more than 30 years. Rapid clearance of the enzyme from blood stream and its L-glutaminase-dependent neurotoxicity has led to searching for new L-asparaginases with more desirable properties. In the present study, L-asparaginase coding gene of Halomonas elongata was isolated, expressed in Escherichia coli, purified, and characterized. The purified protein was found to have a molecular mass of 39.5 kDa and 1000-folds more activity towards L-asparagine than L-glutamine. Enzyme-specific activity towards L-asparagine was determined to be 1510 U/mg, which is among the highest reported values for microbial L-asparaginases. K , V, and k values were 5.6 mM, 2.2 μmol/min, and 1.96 × 10 1/S, respectively. Optimum temperature was found to be 37 °C while the enzyme showed maximum activity at a wide pH range (from 6 to 9). Enzyme half-life in the presence of human serum at 37 °C was 90 min which is three times higher when compared with reported values for E. coli L-asparaginase. Enzyme showed cytotoxic effects against Jurkat and U937 cell lines with an IC of 2 and 1 U/ml, respectively. Also, no toxic effects on human erythrocytes and Chinese hamster ovary cell lines were detected, and just minor inhibitory effects on human umbilical vein endothelial cells were observed. This is the first report describing the therapeutic potentials of a recombinant L-asparaginase isolated from a halophilic bacterium as an anticancer agent.
L-天冬酰胺酶已在治疗急性淋巴细胞白血病（ALL）患者中使用超过 30 年。由于该酶在血液中快速清除以及其依赖于 L-谷氨酰胺的神经毒性，人们一直在寻找具有更理想特性的新型 L-天冬酰胺酶。本研究中，分离了盐单胞菌 Halomonas elongata 的 L-天冬酰胺酶编码基因，在大肠杆菌中表达、纯化并进行了表征。该纯化蛋白的分子量为 39.5 kDa，对 L-天冬酰胺的活性比 L-谷氨酰胺高 1000 倍。对 L-天冬酰胺的酶比活为 1510 U/mg，属于微生物 L-天冬酰胺酶的最高报道值之一。K 、V 和 k 值分别为 5.6 mM、2.2 μmol/min 和 1.96×10 1/S。最适温度为 37°C，而该酶在较宽的 pH 范围内（6 至 9）显示最大活性。在 37°C 下，人血清中酶的半衰期为 90 分钟，与报道的大肠杆菌 L-天冬酰胺酶相比，半衰期提高了三倍。该酶对 Jurkat 和 U937 细胞系表现出细胞毒性作用，IC 分别为 2 和 1 U/ml。同时，未检测到对人红细胞和中国仓鼠卵巢细胞系的毒性作用，仅观察到对人脐静脉内皮细胞的轻微抑制作用。这是首次报道从嗜盐菌中分离的重组 L-天冬酰胺酶作为抗癌剂的治疗潜力。
