Combining binding pocket mutagenesis and substrate tunnel engineering to improve an (R)-selective transaminase for the efficient synthesis of (R)-3-aminobutanol

( R )-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven ( R )-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi , virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency ( k cat / K m ) than the wild-type enzyme . Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of ( R )-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of ( R )-3-aminobutanol. • Seven potential novel ( R )-selective transaminases were successfully characterized by homologous sequence mining. • Virtual mutagenesis and substrate tunnel engineering for enhancing enzyme activity were successfully implemented. • A variant T282S/Q137E performing 3.68-fold greater catalytic efficiency ( k cat / K m ) was successfully constructed. • The variant achieved a 93 % conversion of 100 mM 4-hydroxy-2-butanone through substrate fed-batch and air sweeping processes.