Enhanced L-theanine production through semi-rational design of γ-glutamylmethylamide synthetase from Methylovorus mays

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Enzyme and Microbial Technology Pub Date : 2024-07-17 DOI:10.1016/j.enzmictec.2024.110481

Chao Fan , Jiakun Qi , Yunhan Cong , Chunzhi Zhang

引用次数: 0

Abstract

The thermal instability of γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays has imposed limitations on its industrial applications, affecting both stability and activity at reaction temperatures. In this study, disulfide bridges were introduced through a combination of directed evolution and rational design to enhance GMAS stability. Among the variants that we generated, M12 exhibited a 1.46-fold improvement in relative enzyme activity and a 6.23-fold increase in half-life at 40℃ compared to the wild-type GMAS. Employing variant M12 under optimal conditions, we achieved the production of 645.7 mM (112.49 g/L) L-theanine with a productivity of 29.3 mM/h, from 800 mM substrate in an ATP regeneration system. Our strategy significantly enhances the biosynthesis efficiency of L-theanine by preserving the structural stability of the enzyme during the catalysis process.

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通过对 Methylovorus mays 的 γ-谷氨酰甲酰胺合成酶进行半合理设计，提高 L-茶氨酸的产量。

来自Methylovorus mays的γ-谷氨酰甲酰胺合成酶（GMAS）的热不稳定性限制了其工业应用，影响了其在反应温度下的稳定性和活性。本研究通过定向进化和合理设计相结合的方法引入了二硫桥，以提高 GMAS 的稳定性。在我们生成的变体中，M12与野生型GMAS相比，相对酶活性提高了1.46倍，在40℃时的半衰期延长了6.23倍。在最佳条件下使用变体 M12，我们在 ATP 再生系统中以 29.3 mM/h 的生产率从 800 mM 底物生产出了 645.7 mM（112.49 g/L）L-茶氨酸。我们的策略在催化过程中保持了酶的结构稳定性，从而大大提高了 L-茶氨酸的生物合成效率。

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

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.