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Efficient preparation of c-di-AMP at gram-scale using an immobilized Vibrio cholerae dinucleotide cyclase DncV
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2021-02-01 , DOI: 10.1016/j.enzmictec.2020.109700
Qichao Sun 1 , Yun Lv 1 , Chenhui Zhang 2 , Weifang Wu 1 , Rui Zhang 3 , Chunyuan Zhu 4 , Yao-Yao Li 2 , Huiqing Yuan 1 , Jing Zhu 5 , Deyu Zhu 1
Affiliation  

Cyclic di-AMP is a bacterial nucleotide second messenger and evaluated as a potential vaccine adjuvant candidate. Here, we report a practical and economical enzymatic method for gram-scale preparation of c-di-AMP using an immobilized Vibrio cholerae dinucleotide cyclase DncV. The method mainly includes four steps: preparation of DncV-immobilized resin, enzymatic synthesis of c-di-AMP, purification using macroporous absorption resin SP207, and desiccation using rotary evaporation and lyophilization. Enzymatic synthesis is the most critical step, and almost all substrate ATP was converted to c-di-AMP under an optimum condition in which 300 mL of 300 mM NH4Ac/NH3 pH 9.5 buffer supplemented with 20 mM MnCl2, 10 mM ATP and 4 mL of DncV-immobilized resin containing ∼19 mg DncV were incubated at 30 °C overnight. After purification, up to 1 g of the diammonium salt of c-di-AMP with weight purity of ≥98% was obtained as white powder, which corresponds to an overall yield of ∼80% based on the ATP input into the reaction. The method is easily performed in laboratory to prepare c-di-AMP on a gram scale and could be used in industry on a large scale.

中文翻译:

使用固定化霍乱弧菌二核苷酸环化酶 DncV 高效制备克级 c-di-AMP

Cyclic di-AMP 是一种细菌核苷酸第二信使,被评估为潜在的疫苗佐剂候选物。在这里,我们报告了一种使用固定化霍乱弧菌二核苷酸环化酶 DncV 进行克级规模制备 c-di-AMP 的实用且经济的酶促方法。该方法主要包括四个步骤:DncV固定化树脂的制备、c-di-AMP的酶法合成、大孔吸附树脂SP207纯化、旋转蒸发和冻干干燥。酶促合成是最关键的步骤,在 300 mL 300 mM NH4Ac/NH3 pH 9.5 缓冲液中添加 20 mM MnCl2、10 mM ATP 和 4 mL 的最佳条件下,几乎所有底物 ATP 都转化为 c-di-AMP将含有约 19 mg DncV 的 DncV 固定化树脂在 30 °C 下孵育过夜。净化后,获得高达 1 g 重量纯度≥98% 的 c-di-AMP 的二铵盐,为白色粉末,基于进入反应的 ATP 输入,这对应于约 80% 的总产率。该方法很容易在实验室中进行,以克规模制备 c-di-AMP,并可在工业中大规模使用。
更新日期:2021-02-01
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