Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis
Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis
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DOI：
10.3390/molecules21121621
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发表时间：
2016-12-01
期刊：
影响因子：
4.6
通讯作者：
Terreni, Marco
中科院分区：
文献类型：
--
作者：
Bavaro, Teodora;Cattaneo, Giulia;Terreni, Marco
本文介绍了枯草芽孢杆菌中性蛋白酶的固定化及其在乙酰化核苷的区域选择性水解中的应用，包括用于制备抗癌产品的构建块。关于固定化研究，不同的固定化方法得到了不同的结果。在要求的反应条件下，环氧疏水载体提供了一种稳定性差的衍生物，几乎释放了50%的固定化蛋白质。相反，在不同活化的亲水性载体（琼脂糖）上的共价固定产生了非常稳定的酶衍生物。为了解释获得的酶固定化结果，以金黄色葡萄球菌金属蛋白酶的结构为模板，在硅基构建枯草芽孢杆菌蛋白酶N的三维结构模型中预测了赖氨酸在酶表面的假设定位。该固定化酶对不同过乙酰化核苷的水解具有较高的区域选择性。获得了一种稳定的酶衍生物，并成功地用于开发乙酰化核苷的高效制备生物工艺，为高收率合成卡培他滨提供了新的中间体。
This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield.