N-methyl-d-aspartic acid (NMDA), an amino acid existing in human and animal central nervous system, exerts agonist action on one of the glutamate receptor subtypes and is clinically used for treatment of diabetes, Parkinson’s and Alzheimer’s syndrome. In this study, an enzymatic protocol for the chiral resolution of N-methyl-d,l-aspartic acid was built with a predicted N-demethylase (GenBank ID: OJV90073.1) from the genome of Chloroflexi bacterium 54-19. Through sequence alignment, the enzyme shares an identity of 32.19% to 2UZZ (PDB ID) with a conserved catalytic center. Recombinantly expressed in Bacillus subtilis WB600, the N-demethylase was characterized with optimal temperature and pH at 55 ℃ and 7.5, and adaptive temperature and pH were 40–60 ℃ and 6–8. The effects of organic solvents and metal ions were investigated as well. Compared with other previously reported sarcosine oxidases, the enzyme showed a specific N-demethylation activity against N-methyl-l-aspartic acid according to the analysis by chiral liquid chromatography, LC–MS/MS and a detection by polarimeter. The results demonstrated 76% of 4.5 mM N-methyl-d,l-aspartic acid could be chirally separated by 7 mg·L⁻¹ enzyme after reaction of 80 min. This work provided a foundation for mild synthesis of NMDA in industry.

Graphical abstract

中文翻译：

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使用来自 Chloroflexi 细菌 54-19 的预测 N-去甲基酶对 N-甲基-d,l-天冬氨酸进行手性拆分

N-甲基-d-天冬氨酸(NMDA)是一种存在于人和动物中枢神经系统中的氨基酸，对其中一种谷氨酸受体亚型发挥激动作用，临床上用于治疗糖尿病、帕金森氏症和阿尔茨海默氏症。在本研究中，使用来自Chloroflexi 细菌54-19 基因组的预测N-去甲基化酶（GenBank ID：OJV90073.1）构建了用于手性拆分N-甲基-d，l-天冬氨酸的酶促方案。通过序列比对，该酶与具有保守催化中心的 2UZZ (PDB ID) 具有 32.19% 的同一性。在枯草芽孢杆菌WB600中重组表达，N-去甲基化酶的最适温度和pH为55℃和7.5，适应温度和pH分别为40～60℃和6～8。还研究了有机溶剂和金属离子的影响。与其他先前报道的肌氨酸氧化酶相比，根据手性液相色谱分析、LC-MS/MS 和旋光仪检测，该酶显示出对N-甲基-L-天冬氨酸的特异性N-去甲基化活性。结果表明，7 mg·L ^-1酶反应80 min后，76%的4.5 mM N-甲基-d , l-天冬氨酸可手性分离。该工作为工业上温和合成NMDA奠定了基础。

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