Development of an enzyme activity screening system for β-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting
Development of an enzyme activity screening system for β-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting
复制标题
DOI：
10.1007/s00253-009-2091-8
复制
发表时间：
2009-06
影响因子：
5
通讯作者：
Yan Zhou;S. Kajiyama;Kouichi Itoh;Takanori Tanino;Nobuo Fukuda;Tsutomu Tanaka;A. Kondo;K. Fukui
中科院分区：
文献类型：
--
作者：
Yan Zhou;S. Kajiyama;Kouichi Itoh;Takanori Tanino;Nobuo Fukuda;Tsutomu Tanaka;A. Kondo;K. Fukui
最近关于使用流式细胞术对酵母细胞进行高速亲和筛选系统的报道尚未适用于筛选展示水解酶的酵母细胞，因为从荧光素底物释放的荧光分子在酶促反应后扩散到溶液中。在这项研究中，通过使用新开发的反胶束方法，将展示β-糖苷酶的酵母细胞单独捕获在微型海藻酸钙珠中，以防止水解荧光底物的扩散。通过对这些捕获的细胞进行流式分选，活性细胞从与阴性对照的混合悬浮液中成功富集约 82 倍。该系统应该是高速筛选展示各种水解酶的酵母细胞的有用方法，并且具有筛选具有更高活性的酶展示酵母细胞的随机文库的潜在应用。
Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying β-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.