Fluorogenic substrates for the screening assay of transketolase through beta-elimination of umbelliferone-Development, scope and limitations

被引：5
作者：
Charmantray, F. [1 ,2 ]
Legeret, B. [1 ,2 ]
Helaine, V. [1 ,2 ]
Hecquet, L. [1 ,2 ]
机构：
[1] CNRS, SEESIB, UMR6504, F-63177 Aubiere, France
[2] Univ Clermont Ferrand, Univ Blaise Pascal, Lab SEESIB, F-63000 Clermont Ferrand, France
来源：
JOURNAL OF BIOTECHNOLOGY | 2010年 / 145卷 / 04期
关键词：
Biocatalysis; Fluorogenic substrate; beta-Elimination; High-throughput screening; Transketolase; C-C bond; SACCHAROMYCES-CEREVISIAE; STEREOCHEMICAL PROBES; ENZYMATIC-SYNTHESIS; ALDOLASES; ISOMERIZATION; MECHANISM; CATALYSIS; ESTERASES; ENZYMES; LIPASES;
D O I：
10.1016/j.jbiotec.2009.12.022
中图分类号：
Q81 [生物工程学（生物技术）]; Q93 [微生物学];
学科分类号：
071005 ; 0836 ; 090102 ; 100705 ;
摘要：
5-O-Coumarinyl-D-xylulose was studied as a fluorogenic substrate for the stereospecific assay of transketolase enzyme. Enzymatic C2-C3 cleavage released an alpha-hydroxyl, beta-coumarinyl substituted aldehyde. Although the subsequent P-elimination step was rate limiting under chemical or enzymatic catalysis, we detected a TK activity as low as 0.7 mlU. To improve the fluorescence signal release, kinetic and product distribution analyses of this reaction were performed by LC/UV/MS coupling. (C) 2010 Elsevier B.V. All rights reserved.
引用
收藏
页码：359 / 366
页数：8
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