Marine Biotechnology Division, National Institute of Ocean technology, Pallikaranai, Chennai, 600100, Tamilnadu, India.
Department of Chemical Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, Republic of Korea.
Sci Rep. 2024 Nov 4;14(1):26676. doi: 10.1038/s41598-024-77959-6.
An alkaline active L-glutaminase (BALG) producing bacterium was screened and identified from seamount sediment samples of the Arabian Sea. The isolate was confirmed to be Bacillus australimaris NIOT30 based on morphological characteristics and 16 S rRNA gene sequencing. The glutaminase gene, balg was PCR amplified, cloned and expressed in E. coli BL21 (DE3) host. The molecular weight of purified BALG was estimated to be 36 kDa and the enzyme showed a specific activity of 507 ± 27 Umg against L-glutamine under optimal assay conditions of pH 7.0 and temperature at 37 °C for 15 min. The enzyme showed maximum activity at pH 7 and retained 95% activity at pH 10. BALG retained a relative activity of about 82% and 45% at 45 °C and 60 °C respectively. The kinetic parameters of BALG, Km and Kcat/Km were determined to be of 210 ± 11 mM and 4.4 × 10 M s respectively. Homology modeling and substrate ligand interaction studies revealed the stability of the enzyme-substrate complex. The present study highlights the characterization of a highly active L-glutaminase from B. australimaris NIOT30. Further, mutational analyses of ligand binding residues would show insights into the affinity of L-Glutaminase.
从阿拉伯海海山沉积物样本中筛选和鉴定出一种产碱性活性 L-谷氨酰胺酶（BALG）的细菌。根据形态特征和 16S rRNA 基因测序，该分离物被确认为芽孢杆菌属澳大利亚亚种 NIOT30。通过 PCR 扩增、克隆和在大肠杆菌 BL21(DE3)宿主中表达，扩增、克隆和表达了谷氨酰胺酶基因 balg。纯化的 BALG 的分子量估计为 36 kDa，在最佳测定条件下（pH 7.0，温度 37°C，15 min），该酶对 L-谷氨酰胺的比活性为 507±27 Umg。该酶在 pH 7 时表现出最大活性，在 pH 10 时保留 95%的活性。BALG 在 45°C 和 60°C 下分别保留约 82%和 45%的相对活性。BALG 的动力学参数 Km 和 Kcat/Km 分别确定为 210±11 mM 和 4.4×10 M s。同源建模和底物配体相互作用研究揭示了酶-底物复合物的稳定性。本研究强调了从澳大利亚芽孢杆菌 NIOT30 中鉴定出一种高活性的 L-谷氨酰胺酶的特性。此外，配体结合残基的突变分析将揭示 L-谷氨酰胺酶的亲和力。
