Enantioselective Resolution of β‐ and γ‐Lactams by a Single Amidase From <scp> <i>Rhodococcus baikonurensis</i> </scp> Enables Efficient Access to Key Intermediates for Paclitaxel and Carbocyclic Nucleosides

Enantiomerically pure β- and γ-lactams are vital chiral building blocks widely used in the synthesis of antiviral and anticancer drugs. However, producing them on a large scale through chemical synthesis and enantiomeric separation is both expensive and harmful to the environment. Using integrative bioinformatics, we identified a new signature amidase, Rb-Ami, from the haloalkaliphilic bacterium Rhodococcus baikonurensis. The Rb-ami gene was cloned and successful expressed in Escherichia coli BL21. Soluble and active recombinant enzyme was obtained, and in vitro studies show that this enzyme can efficiently catalyze the enantioselective hydrolysis of different lactam substrates. Specifically, Rb-Ami can effectively resolve racemic mixtures of 3-hydroxy-4-phenylazetidin-2-one (β-lactam) and 2-azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam), delivering enantiomeric excesses greater than 99.0% and yields above 49.0%. To our knowledge, this is the first reported amidase exhibiting both γ-lactamase and β-lactamase activities, and the first β-lactamase used for the kinetic resolution of racemic 3-hydroxy-4-phenylazetidin-2-one. This enzymatic method provides a highly efficient and eco-friendly approach for producing the paclitaxel side chain and precursors of carbocyclic nucleoside antivirals.