Integrated production and protein-protein docking analysis of L-methionase from Klebsiella oxytoca for enzyme-based anticancer therapy via methionine starvation and epigenetic modulation.

IF 2.9 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
3 Biotech Pub Date : 2025-10-01 Epub Date: 2025-09-09 DOI:10.1007/s13205-025-04501-4
Bhupender Sharma, Vivek Chauhan, Vivek Kumar Dhiman, Rakesh Kumar, Gaytri Mahajan, Sukhdev Singh, Kanta Sashi, Anjali Kashwal, Shamsher S Kanwar
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引用次数: 0

Abstract

Intracellular L-methionine γ-lyase (MGL) from Klebsiella oxytoca BLM-1 was produced and optimized using a combination of One-Factor-at-a-Time and Response Surface Methodology. Optimal culture conditions, such as pH 9.0, 3% (w/v) lactose, and 1.02% (w/v) malt extract, resulted in the highest intracellular MGL activity (0.235 U/mL), representing a 1.13-fold improvement over initial conditions, with a total yield of 40.80 U from a 2 L optimized broth. Purification using Octyl-Sepharose chromatography produced a highly active multimeric enzyme (~ 250 kDa) with 0.384 U/mL activity, which was confirmed as a heteromeric complex (~ 63 kDa and ~ 117 kDa subunits) by SDS-PAGE. The enzyme displayed strong cytotoxic activity toward methionine-dependent cancer cell lines, with IC₅₀ values of 0.023 U for HepG2 and 0.0045 U for A549, while exerting minimal effects on HEK-293 cells. Molecular docking revealed that L-methionine binds to the MGL active site with a binding energy of - 6.5 kcal/mol, and protein-protein docking identified favorable interactions with key methionine pathway enzymes, including METAP2 (- 902.3), MAT2A (- 895.1), and SAHH (- 890.9). These findings highlight the successful optimization of MGL production, its effective purification, and its significant anticancer potential, providing a strong foundation for its development as a therapeutic enzyme for methionine-dependent tumors.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04501-4.

氧化克雷伯菌l -蛋氨酸酶的集成生产和蛋白对接分析,通过蛋氨酸饥饿和表观遗传调控进行酶基抗癌治疗。
利用单因子法和响应面法对产自产氧克雷伯菌BLM-1的细胞内l -蛋氨酸γ-裂解酶(MGL)进行了优化。最佳培养条件为pH 9.0,3% (w/v)乳糖和1.02% (w/v)麦芽提取物,细胞内MGL活性最高(0.235 U/mL),比初始条件提高1.13倍,2 L优化肉汤的总产量为40.80 U。采用辛基- sepharose层析纯化得到了一个高活性的多聚酶(~ 250 kDa),活性0.384 U/mL, SDS-PAGE证实该多聚酶为异聚物(~ 63 kDa和~ 117 kDa亚基)。该酶对依赖蛋氨酸的癌细胞系显示出很强的细胞毒活性,对HepG2和A549的IC₅0值分别为0.023 U和0.0045 U,而对HEK-293细胞的影响最小。分子对接发现l -蛋氨酸结合到MGL活性位点的结合能为- 6.5 kcal/mol,蛋白-蛋白对接发现了与关键蛋氨酸途径酶的良好相互作用,包括METAP2(- 902.3)、MAT2A(- 895.1)和SAHH(- 890.9)。这些发现突出了MGL生产的成功优化,其有效的纯化及其显著的抗癌潜力,为其作为蛋氨酸依赖肿瘤的治疗酶的发展提供了坚实的基础。补充信息:在线版本包含补充资料,提供地址为10.1007/s13205-025-04501-4。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
3 Biotech
3 Biotech Agricultural and Biological Sciences-Agricultural and Biological Sciences (miscellaneous)
CiteScore
6.00
自引率
0.00%
发文量
314
期刊介绍: 3 Biotech publishes the results of the latest research related to the study and application of biotechnology to: - Medicine and Biomedical Sciences - Agriculture - The Environment The focus on these three technology sectors recognizes that complete Biotechnology applications often require a combination of techniques. 3 Biotech not only presents the latest developments in biotechnology but also addresses the problems and benefits of integrating a variety of techniques for a particular application. 3 Biotech will appeal to scientists and engineers in both academia and industry focused on the safe and efficient application of Biotechnology to Medicine, Agriculture and the Environment.
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