Seo Dong-Ho, Jung Jong-Hyun, Park Cheon-Seok
Research Group of Gut Microbiome, Korea Food Research Institute, Seongnam 13539, South Korea; Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 17104, South Korea.
Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 56212, South Korea; Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 17104, South Korea.
Anal Biochem. 2017 Sep 1;532:19-25. doi: 10.1016/j.ab.2017.05.028. Epub 2017 May 31.
The purpose of this study was to investigate the novel fluorescence-based assay for the transglycosylation activity of amylosucrase (ASase). The transglycosylation activity of ASase from Deinococcus geothermalis (DGAS), ASase from Neisseria polysaccharea (NPAS), and DGAS-B (chimeric ASase wherein the B domain from DGAS was exchanged with the B domain of NPAS in a DGAS background) was applied to modify 4-methlylumberlliferone (MU) to 4-methylumberlliferone glucoside (MUG) using MU as an acceptor and sucrose as a glucoside donor. The result of HPLC (high performance liquid chromatography) show that the bioconversion of MUG with ASases was successfully accomplished using sucrose and MU. Kinetic studies of ASases were performed to determine kinetic parameter for sucrose and MU. The order of overall performance (k/K) of transglycosylation activity for MU among DGAS, DGAS-B and NPAS was as follows: DGAS-B (8.1) > DGAS (5.0) > NPAS (0.4). The fluorescence-based transglycosylation assay using MU has a potential to be used as the detection of transglycosylation activity of ASase and to screen novel ASase variants, which may be improved in their transglycosylation activities.
本研究的目的是探究用于测定淀粉蔗糖酶（ASase）转糖基化活性的新型荧光检测法。利用来自嗜热栖热放线菌的ASase（DGAS）、来自多糖奈瑟菌的ASase（NPAS）以及DGAS-B（一种嵌合ASase，其中DGAS的B结构域在DGAS背景下与NPAS的B结构域进行了交换）的转糖基化活性，以4-甲基伞形酮（MU）为受体、蔗糖为糖苷供体，将4-甲基伞形酮转化为4-甲基伞形酮葡萄糖苷（MUG）。高效液相色谱（HPLC）结果表明，使用蔗糖和MU，ASases成功实现了MUG的生物转化。对ASases进行动力学研究以确定蔗糖和MU的动力学参数。DGAS、DGAS-B和NPAS中MU转糖基化活性的总体性能顺序（k/K）如下：DGAS-B（8.1）>DGAS（5.0）>NPAS（0.4）。使用MU的基于荧光的转糖基化检测法有潜力用于检测ASase的转糖基化活性并筛选新型ASase变体，这些变体的转糖基化活性可能会得到改善。
