کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5131735 1491329 2017 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A high-throughput microtiter plate assay for the discovery of active and enantioselective amino alcohol-specific transaminases
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
A high-throughput microtiter plate assay for the discovery of active and enantioselective amino alcohol-specific transaminases
چکیده انگلیسی

Chiral vicinal amino alcohols are important chiral building blocks and intermediates in the pharmaceutical industry. The transaminase (TAm) catalyzed kinetic resolution of racemic amino alcohols provides a straightforward approach to access these important compounds. This study describes the development of a novel microtiter plate assay to screen vicinal amino alcohol-specific TAms using a tetrazolium red-based colorimetric assay to monitor the rate of α-hydroxy ketone formation at 510 nm. This approach is the first to determine the Michaelis-Menten parameters for a recombinant TAm (PpbauA) from Pseudomonas putida NBRC14164. The corresponding Vmax and KM values for both enantiomers of 2-amino-1-propanol and 2-amino-1-butanol were obtained, and the calculated kinetic E-factors of PpbauA toward 2-amino-1-propanol and 2-amino-1-butanol are 3 (S) and 6 (R), respectively. The method is sensitive and exhibits low level background coloration. Moreover, this method can be used to detect transaminase activity and enantioselectivity toward amino alcohols in a high-throughput format. Additionally, this simple method is compatible with the most widely used (R)- and (S)-selective transaminases and may be a broadly applicable tool for screening transaminases from a transaminase mutant library.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 518, 1 February 2017, Pages 94-101
نویسندگان
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