Fujii Takahiro, Igarashi Kiyohiko, Samejima Masahiro
1 Ikeda Food Research Co., Ltd.
2 Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo.
J Appl Glycosci (1999). 2020 Sep 3;67(3):73-78. doi: 10.5458/jag.jag.JAG-2020_0002. eCollection 2020.
Pyranose 2-oxidases catalyze the oxidation of various pyranose sugars at the C2 position. However, their potential application for detecting sugars other than glucose in blood is hindered by relatively high activity towards glucose. In this study, in order to find a mutant enzyme with enhanced specificity for 1,5-anhydro-D-glucitol (1,5-AG), which is a biomarker for diabetes mellitus, we conducted site-directed mutagenesis of pyranose 2-oxidase from the basidiomycete ( POX). Considering the three-dimensional structure of the substrate-binding site of POX and the structural difference between glucose and 1,5-AG, we selected alanine 551 of POX as a target residue for mutation. Kinetic studies of the 19 mutants of POX expressed as recombinant proteins in revealed that the ratio of / for 1,5-AG to / for glucose was three times higher for the A551L mutant than for wild-type POX. Although the A551L mutant has lower specific activity towards each substrate than the wild-type enzyme, its increased specificity for 1,5-AG makes it a promising lead for the development of POX-based 1,5-AG detection systems.
吡喃糖2-氧化酶催化各种吡喃糖在C2位的氧化反应。然而，它们对血液中除葡萄糖以外的糖类进行检测的潜在应用受到其对葡萄糖相对较高活性的阻碍。在本研究中，为了找到一种对1,5-脱水-D-葡萄糖醇（1,5-AG）具有更高特异性的突变酶，1,5-AG是糖尿病的一种生物标志物，我们对担子菌的吡喃糖2-氧化酶（POX）进行了定点诱变。考虑到POX底物结合位点的三维结构以及葡萄糖和1,5-AG之间的结构差异，我们选择POX的丙氨酸551作为突变的目标残基。对在中作为重组蛋白表达的POX的19个突变体进行的动力学研究表明，A551L突变体对1,5-AG的/比值与对葡萄糖的/比值之比是野生型POX的三倍。尽管A551L突变体对每种底物的比活性低于野生型酶，但其对1,5-AG增加的特异性使其成为开发基于POX的1,5-AG检测系统的一个有前景的先导物。
