Kumar Subhash, Darnal Sanyukta, Patial Vijeta, Kumar Virender, Kumar Vijay, Kumar Sanjay, Singh Dharam
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176 061 India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201 002 India.
3 Biotech. 2022 Aug;12(8):162. doi: 10.1007/s13205-022-03224-0. Epub 2022 Jul 9.
l-Asparaginase (l-ASNase) is a key enzyme used to treat acute lymphoblastic leukemia, a childhood blood cancer. Here, we report on the characterization of a recombinant l-ASNase (44- II) from sp. PCH44. The gene was identified from its genome, cloned, and overexpressed in the host (). The recombinant l-ASNase (44-ASNase II) was purified with a monomer size of 37.0 kDa and a homotetrameric size of 148.0 kDa. The purified 44-ASNase II exhibited optimum activity of 40.84 U/mg in Tris-HCl buffer (50 mM, pH 8.5) at 45 °C for 15 min. It retained 76.53% of enzyme activity at 45 °C after 120 min of incubation. The half-life and values were 600 min and 1.10 × 10 min, respectively, at 45 °C. The kinetic constants values and were 0.56, 0.728 mM, and 29.41, 50.12 U/mg for l-asparagine and l-glutamine, respectively. However, for l-glutamine is more (30.91 s) than l-asparagine (18.06 s), suggesting that enzymes act more efficiently on l-glutamine than l-asparagine. The docking analysis of l-asparagine and l-glutamine with active site residues of the enzyme revealed a molecular basis for high l-glutaminase (L-GLNase) activity and provided insights into the role of key amino acid residues in the preferential enzymatic activities.
The online version contains supplementary material available at 10.1007/s13205-022-03224-0.
L-天冬酰胺酶（L-ASNase）是用于治疗急性淋巴细胞白血病（一种儿童血癌）的关键酶。在此，我们报告了来自sp. PCH44的重组L-天冬酰胺酶（44-II）的特性。该基因从其基因组中鉴定出来，进行克隆，并在宿主（）中过表达。重组L-天冬酰胺酶（44-ASNase II）经纯化后，单体大小为37.0 kDa，同四聚体大小为148.0 kDa。纯化后的44-ASNase II在45°C的Tris-HCl缓冲液（50 mM，pH 8.5）中孵育15分钟时，表现出40.84 U/mg的最佳活性。在45°C孵育120分钟后，它保留了76.53%的酶活性。在45°C时，半衰期和值分别为600分钟和1.10×10分钟。L-天冬酰胺和L-谷氨酰胺的动力学常数分别为0.56、0.728 mM和29.41、50.12 U/mg。然而，L-谷氨酰胺的（30.91秒）比L-天冬酰胺的（18.06秒）更长，这表明该酶对L-谷氨酰胺的作用比对L-天冬酰胺更有效。L-天冬酰胺和L-谷氨酰胺与该酶活性位点残基的对接分析揭示了高L-谷氨酰胺酶（L-GLNase）活性的分子基础，并为关键氨基酸残基在优先酶促活性中的作用提供了见解。
在线版本包含可在10.1007/s13205-022-03224-0获取的补充材料。
