Active Pocket Remodeling Coupled with Substrate Tunnel Engineering of ω-Transaminase CC1012 for Efficient Synthesis of (S)-1-Boc-3-aminopiperidine

Transaminases provide an effective approach for the asymmetric synthesis of chiral amines. However, native ω-transaminases often suffer from low catalytic efficiency and poor substrate concentration tolerance, limiting industrial applicability. Targeting the synthesis of ( S )-1-Boc-3-aminopiperidine, this study engineered the ( S )-selective ω-transaminase CC1012 from Caulobacter sp. D5 through active pocket remodeling and substrate tunnel engineering. Hydrophobic remodeling of the small binding pocket yielded the single mutant M1 (Q25F) with a 27-fold improvement in intrinsic catalytic efficiency ( k cat / K m ). Conservation analysis and virtual saturation mutagenesis further identified R424 and D320 as key residues in the loop region at the substrate tunnel entrance. Iterative saturation mutagenesis on these sites using M1 as a template produced the optimal mutant M3 (Q25F/D320M/R424L), exhibiting a 45-fold enhancement in k cat / K m over the wild type, achieving 98% conversion with ee > 99.9% at 1000 mM substrate concentration. This study provides a superior biocatalyst for the efficient synthesis of ( S )-1-Boc-3-aminopiperidine.