Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Appl Microbiol Biotechnol. 2022 Sep;106(17):5511-5524. doi: 10.1007/s00253-022-12058-y. Epub 2022 Jul 25.
Because of the therapeutical impacts of hydrolytic enzymes in different diseases, in particular malignancies, we aimed to produce a recombinant putative L-glutaminase (GLS A) from a recently characterized halo-thermotolerant Bacillus sp. SL-1. For this purpose, the glsA gene was identified and efficiently overexpressed in the Origami™ B (DE3) strain. The yield of the purified GLS A was ~ 20 mg/L, indicating a significant expression of recombinant enzyme in the Origami. The enzyme activity assay revealed a significant hydrolytic effect of the recombinant GLS A on L-asparagine (Asn) (i.e., K 39.8 μM, k 19.9 S) with a minimal affinity for L-glutamine (Gln). The GLS A significantly suppressed the growth of leukemic Jurkat cells through apoptosis induction (47.5%) in the IC dosage of the enzyme. The GLS A could also change the Bax/Bcl2 expression ratio, indicating its apoptotic effect on cancer cells. The in silico analysis was conducted to predict structural features related to the histidine-tag exposure in the N- or C-terminal of the recombinant GLS A. In addition, molecular docking simulation for substrate specificity revealed a greater binding affinity of Asn to the enzyme binding-site residues than Gln, which was confirmed in experimental procedures as well. In conclusion, the current study introduced a recombinant GLS A with unique functional and structural features, highlighting its potential pharmaceutical and medical importance. GLS A represents the first annotated enzyme from Bacillus with prominent asparaginase activity, which can be considered for developing alternative enzymes in therapeutic applications. KEY POINTS: • Hydrolytic enzymes have critical applications in different types of human malignancies. • A recombinant L-glutaminase (GLS A) was produced from halo-thermotolerant Bacillus sp. SL-1. • GLS A displayed a marked hydrolytic activity on L-asparagine compared to the L-glutamine. • GLS A with significant substrate promiscuity may be an alternative for developing novel pharmaceuticals.
由于水解酶在各种疾病中的治疗作用，特别是恶性肿瘤，我们旨在从最近鉴定的嗜盐耐热芽孢杆菌 SL-1 中生产一种重组潜在的 L-谷氨酰胺酶（GLS A）。为此，鉴定了 glsA 基因，并在 Origami™ B（DE3）菌株中有效过表达。纯化的 GLS A 的产量约为 20mg/L，表明重组酶在 Origami 中有显著表达。酶活性测定显示重组 GLS A 对 L-天冬酰胺（Asn）（即 K 39.8μM，k 19.9 S）具有显著的水解作用，对 L-谷氨酰胺（Gln）的亲和力极小。GLS A 通过诱导细胞凋亡（酶的 IC 剂量为 47.5%）显著抑制白血病 Jurkat 细胞的生长。GLS A 还可以改变 Bax/Bcl2 表达比率，表明其对癌细胞的凋亡作用。进行了计算机模拟分析以预测重组 GLS A 中 N 或 C 末端组氨酸标签暴露相关的结构特征。此外，对底物特异性的分子对接模拟表明，Asn 与酶结合位点残基的结合亲和力大于 Gln，这在实验过程中也得到了证实。总之，本研究介绍了一种具有独特功能和结构特征的重组 GLS A，突出了其在制药和医学方面的潜在重要性。GLS A 代表了具有显著天冬酰胺酶活性的芽孢杆菌中第一个被注释的酶，可以考虑将其用于治疗应用中的替代酶的开发。关键点：• 水解酶在人类多种恶性肿瘤的治疗中具有重要作用。• 从嗜盐耐热芽孢杆菌 SL-1 中生产了一种重组 L-谷氨酰胺酶（GLS A）。• GLS A 对 L-天冬酰胺的水解活性明显高于 L-谷氨酰胺。• GLS A 具有显著的底物广谱性，可能是开发新型药物的替代物。
