使用荧光团修饰的环糊精直接测定α-淀粉酶

Direct assay for alpha-amylase using fluorophore-modified cyclodextrins.

作者信息

Murayama Takuya, Tanabe Tetsuya, Ikeda Hiroshi, Ueno Akihiko

机构信息

Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-44 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.

出版信息

Bioorg Med Chem. 2006 Jun 1;14(11):3691-6. doi: 10.1016/j.bmc.2006.01.037. Epub 2006 Feb 7.

DOI:10.1016/j.bmc.2006.01.037

PMID:16464599

Abstract

A simple assay method for alpha-amylase was developed based on fluorophore-modified cyclodextrins (CDs). Four kinds of CD derivatives bearing a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety were prepared as artificial substrates for the assay method. The fluorescence intensity of all the NBD-amine-modified CDs decreased upon addition of Aspergillus oryzae alpha-amylase, indicating a reduction in hydrophobicity near the NBD-amine moiety induced by hydrolysis of the CD ring. NC4gammaCD, having a gamma-CD and an amino-tetramethylene spacer, was the most sensitive substrate for the alpha-amylase assay. The initial rate of hydrolysis of NC4gammaCD displayed a liner correlation to the concentration of the alpha-amylase. NC4gammaCD was sensitive to the alpha-amylase but was not sensitive to guest compounds that were accommodated by the native CDs.

摘要

基于荧光团修饰的环糊精（CDs）开发了一种简单的α-淀粉酶测定方法。制备了四种带有4-氨基-7-硝基苯并-2-恶唑-1,3-二氮唑（NBD-胺）部分的CD衍生物作为该测定方法的人工底物。加入米曲霉α-淀粉酶后，所有NBD-胺修饰的CDs的荧光强度均降低，表明CD环水解导致NBD-胺部分附近的疏水性降低。具有γ-环糊精和氨基四亚甲基间隔基的NC4γCD是α-淀粉酶测定中最敏感的底物。NC4γCD的初始水解速率与α-淀粉酶浓度呈线性相关。NC4γCD对α-淀粉酶敏感，但对天然CDs容纳的客体化合物不敏感。