Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City, 60004, Taiwan.
Institute of Molecular Biology, Academia Sinica, Nangang District, Taipei City, 11529, Taiwan.
Appl Microbiol Biotechnol. 2022 Mar;106(5-6):1991-2006. doi: 10.1007/s00253-022-11836-y. Epub 2022 Mar 1.
The regulation of enzyme activity through complexation with certain metal ions plays an important role in many biological processes. In addition to divalent metals, monovalent cations (MVCs) frequently function as promoters for efficient biocatalysis. Here, we examined the effect of MVCs on the enzymatic catalysis of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis ATCC 27,811 and the application of a metal-activated enzyme to L-theanine synthesis. The transpeptidase activity of BlrGGT was enhanced by Cs and Na over a broad range of concentrations with a maximum of 200 mM. The activation was essentially independent of the ionic radius, but K contributed the least to enhancing the catalytic efficiency. The secondary structure of BlrGGT remained mostly unchanged in the presence of different concentrations of MVCs, but there was a significant change in its tertiary structure under the same conditions. Compared with the control, the half-life (t) of the Cs-enriched enzyme at 60 and 65 °C was shown to increase from 16.3 and 4.0 min to 74.5 and 14.3 min, respectively. The simultaneous addition of Cs and Mg ions exerted a synergistic effect on the activation of BlrGGT. This was adequately reflected by an improvement in the conversion of substrates to L-theanine by 3.3-15.1% upon the addition of 200 mM MgCl into a reaction mixture comprising the freshly desalted enzyme (25 μg/mL), 250 mM L-glutamine, 600 mM ethylamine, 200 mM each of the MVCs, and 50 mM borate buffer (pH 10.5). Taken together, our results provide interesting insights into the complexation of MVCs with BlrGGT and can therefore be potentially useful to the biocatalytic production of naturally occurring γ-glutamyl compounds. KEY POINTS: • The transpeptidase activity of B. licheniformis γ-glutamyltranspeptidase can be activated by monovalent cations. • The thermal stability of the enzyme was profoundly increased in the presence of 200 mM Cs. • The simultaneous addition of Csand Mgions to the reaction mixture improves L-theanine production.
通过与某些金属离子形成复合物来调节酶活性在许多生物过程中起着重要作用。除了二价金属离子外，单价阳离子（MVCs）通常也作为有效的生物催化促进剂。在这里，我们研究了 MVCs 对重组 γ-谷氨酰转肽酶（来自地衣芽孢杆菌 ATCC 27,811 的 BlrGGT）的酶催化作用的影响，以及将金属激活酶应用于茶氨酸合成。BlrGGT 的转肽酶活性在很宽的浓度范围内被 Cs 和 Na 增强，最大浓度为 200 mM。这种激活基本上与离子半径无关，但 K 对提高催化效率的贡献最小。不同浓度的 MVCs 存在时，BlrGGT 的二级结构基本保持不变，但在相同条件下其三级结构有显著变化。与对照相比，在 60 和 65°C 下，富含 Cs 的酶的半衰期（t）分别从 16.3 和 4.0 分钟增加到 74.5 和 14.3 分钟。同时添加 Cs 和 Mg 离子对 BlrGGT 的激活具有协同作用。这在添加 200 mM MgCl 到包含新鲜脱盐酶（25 μg/mL）、250 mM L-谷氨酰胺、600 mM 乙胺、200 mM 每种 MVC 和 50 mM 硼酸盐缓冲液（pH 10.5）的反应混合物中，底物转化为茶氨酸的转化率提高了 3.3-15.1%，这一点得到了充分体现。总之，我们的研究结果为 MVCs 与 BlrGGT 的络合提供了有趣的见解，因此可能对天然 γ-谷氨酰化合物的生物催化生产有用。 关键点： • 地衣芽孢杆菌 γ-谷氨酰转肽酶的转肽酶活性可以被单价阳离子激活。 • 存在 200 mM Cs 时，酶的热稳定性显著提高。 • 向反应混合物中同时添加 Cs 和 Mg 离子可提高茶氨酸产量。
