An enzymic L-2-hydroxyglutarate biosensor based on L-2-hydroxyglutarate dehydrogenase from Azoarcus olearius
An enzymic L-2-hydroxyglutarate biosensor based on L-2-hydroxyglutarate dehydrogenase from Azoarcus olearius
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DOI：
10.1016/j.bios.2023.115740
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发表时间：
2023-10-18
影响因子：
12.6
通讯作者：
Gao，Chao
中科院分区：
文献类型：
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作者：
Hou，Shuang;Kang，Zhaoqi;Gao，Chao
l-2-羟基戊二酸（l-2-HG）是一种关键的信号传导和免疫代谢产物，但其过度积累可导致tol-2-羟基戊二酸尿症、肾癌和其他疾病。迫切需要开发高效、高通量的选择性 1-2-HG 检测方法。本研究从AzoarcusoleariusBH72（AoL2HGDH）的10个同源物中筛选出l-2-HG脱氢酶，并鉴定为对l-2-HG脱氢具有高特异性和活性的酶。然后，开发了一种基于酶法检测的l-2-HG感应荧光报告基因EaLHGFR，其由AoL2HGDH和刃天青组成，用于检测l-2-HG。使用单因素筛选策略系统地优化了 EaLHGFR 的响应幅度和检测限。最佳生物传感器 EaLHGFR-2 的响应幅度为 2189.25 ± 26.89%，检测限为 0.042 μM。它可以准确检测细菌和细胞样品以及人体体液中l-2-HG的浓度。考虑到其理想的特性，EaLHGFR-2 可能是生物样品中 1-2-HG 定量的有前途的替代方案。
l-2-Hydroxyglutarate (l-2-HG) is a critical signaling and immune metabolite but its excessive accumulation can lead tol-2-hydroxyglutaric aciduria, renal cancer, and other diseases. Development of efficient and high-throughput methods for selectivel-2-HG detection is urgently required. In this study,l-2-HG dehydrogenase in AzoarcusoleariusBH72 (AoL2HGDH) was screened from ten homologs and identified as an enzyme with high specificity and activity towardl-2-HG dehydrogenation. Then, anenzymaticassay-basedl-2-HG-sensingfluorescentreporter, EaLHGFR which consists of AoL2HGDH and resazurin, was developed for the detection ofl-2-HG. The response magnitude and limit of detection of EaLHGFR were systematically optimized using a single-factor screening strategy. The optimal biosensor EaLHGFR-2 exhibited a response magnitude of 2189.25 ± 26.89% and a limit of detection of 0.042 μM. It can accurately detect the concentration ofl-2-HG in bacterial and cellular samples as well as human body fluids. Considering its desirable properties, EaLHGFR-2 may be a promising alternative for quantitation ofl-2-HG in biological samples.