Engineering of a carboxylic acid reductase-in the synthesis of pharmaceutical intermediate(S)-2-aminobutanol
Armed with both hydroxyl and amino groups,(S)-2-aminobutanol is an essential chiral building block used in the prepara-tion of diverse pharmaceutical drugs,while effective biocatalysts are highly desired toward the synthesis of(S)-2-aminobutanol.The carboxylic acid reductase derived from Segniliparus rugosus(SrCAR)was used as the model enzyme in the transformation of(S)-2-ami-nobutyric acid to(S)-2-aminobutanol combined with a heterologous alcohol dehydrogenase.After screening the previously constructed SrCAR mutant libraries,the active site was obtained and co-evolution analysis was performed.By using the combinatorial active-site saturation test strategy,the dominant mutant XH7(G430V/E533F/A627N)was obtained.The kcat/Km and Tm values were improved 2.1 fold and 2.3 ℃ compared to that of the wild type enzyme,respectively in the transformation of N-Boc-(S)-2-aminobutyric acid to N-Boc-(S)-2-aminobutyraldehyde.Further,N-Boc-(S)-2-aminobutyraldehyde to the alcohol product can be reduced by the introduc-tion of Pseudomonas fluorescens(PfADH).The bi-enzymatic system containing of XH7 and PfADH enabled a 99％conversion toward 20 mmol/L substrate in 5 h.After deprotection and purification,the yield of(S)-2-aminobutanol reached 60％.Molecular dynamics simulations were performed to shed light on the molecular basis for the improved activity and thermostability of the best mutant.This study expands the biocatalytic toolbox for the synthesis of(S)-2-aminobutanol,and provides guidance for the biosynthesis of similar high value-added pharmaceutical intermediates.
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维普
