A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production
A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production
复制标题
一种控制鞘脂神经酰胺 N-脱酰酶用于溶血鞘糖脂生产反应平衡的简便方法
DOI：
10.1194/jlr.d061176
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发表时间：
2015-09-01
影响因子：
6.5
通讯作者：
Yang, Guang-Yu
中科院分区：
文献类型：
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作者：
Huang, Feng-Tao;Han, Yun-Bin;Yang, Guang-Yu
鞘糖脂(lyso-GSLS)是N-脱酰形式的鞘糖脂(GSLS)，是制备GSL类似物的重要合成中间体。尽管利用鞘磷脂神经酰胺N-脱酰基酶(SCDase)对天然GSLS进行水解制得lyso-GSLS，但由于SCDase还催化逆反应，最终在水解物和合成物之间建立平衡，因此该反应的产率通常较低。在本研究中，我们开发了一种在酶反应体系中引入二价金属阳离子和洗涤剂来控制反应平衡的有效方法。在Ca~(2+)和牛磺酸脱氧胆酸水合物存在下，生成的脂肪酸是通过形成不溶于水的硬脂酸盐而沉淀出来的，并将反应平衡推向水解。GM1水解率最高可达96%，比优化前提高了45%。在制备规模中，从100 mg的GM1中获得了75 mg的Lyso-GM1，产率为90%，这是迄今为止报道的最高产率。该方法还可用于多种GSLS和鞘磷脂的有效水解。因此，该方法应该成为Lyso-GSL生产的一种简便、易扩展和通用的工具，以促进进一步的GSL研究。
Lyso-glycosphingolipids (lyso-GSLs), the N-deacylated forms of glycosphingolipids (GSLs), are important synthetic intermediates for the preparation of GSL analogs. Although lyso-GSLs can be produced by hydrolyzing natural GSLs using sphingolipid ceramide N-deacylase (SCDase), the yield for this reaction is usually low because SCDase also catalyzes the reverse reaction, ultimately establishing an equilibrium between hydrolysis and synthesis. In the present study, we developed an efficient method for controlling the reaction equilibrium by introducing divalent metal cation and detergent in the enzymatic reaction system. In the presence of both Ca2+ and taurodeoxycholate hydrate, the generated fatty acids were precipitated by the formation of insoluble stearate salts and pushing the reaction equilibrium toward hydrolysis. The yield of GM1 hydrolysis can be achieved as high as 96%, with an improvement up to 45% compared with the nonoptimized condition. In preparative scale, 75 mg of lyso-GM1 was obtained from 100 mg of GM1 with a 90% yield, which is the highest reported yield to date. The method can also be used for the efficient hydrolysis of a variety of GSLs and sphingomyelin. Thus, this method should serve as a facile, easily scalable, and general tool for lyso-GSL production to facilitate further GSL research.