Reshaping the substrate binding region of (R)-selective ω-transaminase for asymmetric synthesis of (R)-3-amino-1-butanol.

Appl Microbiol Biotechnol

Jiangsu Key Laboratory of Chiral Pharmaceuticals Biosynthesis, College of Pharmacy and Chemistry & Chemical Engineering, Taizhou University, Taizhou, 225300, Jiangsu, China.

Published: May 2020

(R)-Selective ω-transaminase (ω-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective ω-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a "small-intelligent" library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher k/K value and 9.4 °C higher [Formula: see text] than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective ω-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective ω-TAs.

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Source
http://dx.doi.org/10.1007/s00253-020-10539-6DOI Listing

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