title: A high-throughput microtiter plate assay for the discovery of active and enantioselective amino alcohol-specific transaminases.
abstract: 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 Vmaxand KMvalues 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.
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, PR China. Electronic address: zhangjiandong@tyut.edu.cn.
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Zhang
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, PR China.
authors.forename: Hua-Lei
authors.initials: HL
authors.lastname: Wu
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, PR China.
authors.forename: Tong
authors.initials: T
authors.lastname: Meng
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, PR China. Electronic address: zhangchaofeng@tyut.edu.cn.
authors.forename: Chao-Feng
authors.initials: CF
authors.lastname: Zhang
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, PR China.
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Fan
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, PR China.
authors.forename: Hong-Hong
authors.initials: HH
authors.lastname: Chang
authors.affiliation: Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, PR China.
authors.forename: Wen-Long
authors.initials: WL
authors.lastname: Wei
mesh_terms: D000605:Amino Alcohols / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D011412:Propanolamines / Q000737:chemistry* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity / Q000502:physiology; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000605:Amino Alcohols; D001426:Bacterial Proteins; D011412:Propanolamines; C034448:2-aminopropanol; D000637:Transaminases; C006506:2-amino-1-butanol
keywords: Chiral vicinal amino alcohol; Enantioselective; High-throughput screening; Microtiter plate assay; Transaminase