کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
6451914 1416986 2017 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Development of a colourimetric assay for glycosynthases
ترجمه فارسی عنوان
توسعه یک آزمون رنگی برای گلیکوزینتاز
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
چکیده انگلیسی


- Characterisation of glycosidases (hyperthermophilic, mesophilic, psychrotolerant).
- A colorimetric glycosynthase assay based on a fluoride chemosensor was developed.
- Determination of fluoride concentrations in a MTP format within 10 min.
- The high maximal detection limit allows efficient glycosynthase characterisation.
- Successful application of the assay towards the characterisation of glycosynthases.

The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4 mM and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 257, 10 September 2017, Pages 162-170
نویسندگان
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