Department of Microbiology, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar, Madhya Pradesh 470003, India.
Int J Biol Macromol. 2021 Dec 1;192:28-37. doi: 10.1016/j.ijbiomac.2021.09.186. Epub 2021 Oct 2.
Bacterial L-asparaginase is the key therapeutic enzyme in cancer therapy and is also witnessing demand as a food processing aid. In this study, L-asparaginase of newly isolated Bacillus subtilis ETMC-2 was cloned and over-expressed in Escherichia coli as an active soluble protein using ligation independent cloning strategy. The molecular mass was estimated to be 40 kDa and was optimally active at 50 °C. Zymography revealed that the enzyme was active in homo-tetramer state (~160 KDa). The encoded protein after BLASTp analysis on NCBI showed 99.73% similarity with L-ASNase that of Bacillus sp. Physico-chemical properties were predicted using Protparam leading to categorization of the enzyme as a stable protein with an instability index (II) of 19.02. The calculated aliphatic index (85.44) indicated the high thermal stability of the protein with GRAVY value of -0.317. Protein-Ligand docking revealed that the residues Thr, Thr, and Asp were fundamental in protein-ligand complexation. After homology modelling, model validation was performed using Ramachandran plot, VERIFY3D, and RMSD. The paper describes cloning, heterologous expression, catalytic characteristics and physico-chemical properties of the type II B. subtilis L-ASNase.
细菌 L-天冬酰胺酶是癌症治疗的关键治疗酶，也作为一种食品加工助剂受到需求。在这项研究中，使用连接独立克隆策略，从新分离的枯草芽孢杆菌 ETMC-2 中克隆并在大肠杆菌中过表达了 L-天冬酰胺酶，使其成为一种活性可溶性蛋白。分子量估计为 40 kDa，最适活性温度为 50°C。酶谱分析表明，该酶以同四聚体状态（~160 kDa）发挥活性。经 NCBI 的 BLASTp 分析后，编码蛋白与芽孢杆菌的 L-ASNase 显示出 99.73%的相似性。使用 Protparam 预测理化性质，将该酶归类为稳定蛋白，不稳定性指数（II）为 19.02。计算得出的脂肪指数（85.44）表明该蛋白具有高热稳定性，GRAVY 值为-0.317。蛋白-配体对接表明，残基 Thr、Thr 和 Asp 是蛋白-配体复合物形成的基础。同源建模后，使用 Ramachandran 图、VERIFY3D 和 RMSD 进行模型验证。本文描述了 II 型枯草芽孢杆菌 L-ASNase 的克隆、异源表达、催化特性和理化性质。
