Stereoselective preparation of chiral benzo-γ-lactam by ω-transaminase variants and their immobilized forms using protein crystal as a scaffold

Chiral benzo-γ-lactams are important structural units in many drugs and natural products, yet their biosynthetic strategies remain limited. The ω-transaminase (ω-TABm) from Bacillus megaterium could achieve the biosynthesis of (S)-3-methylisoindolin-1-one (3a) by constructing a one-pot, two-step reaction. Through amine donor optimization, cost-effective isopropylamine (1a) and high conversion-advantaged 4-bromo-α-methylbenzylamine (1n) were identified. Regarding the two amine donors, the excellent variants C61A/L272A and C61W were obtained by semi-rational design, achieving >99.9 % conversions, with enantiomeric excess (e.e._p) of 99.9 % and 91.4 %, respectively, for (S)-3a within 48 h. The stabilities of the variants could be further improved by using protein crystal PhMd41 as a scaffold to realize one-step enzymatic purification and immobilization. The obtained H1-C61A/L272A-PhMd41 and H1-C61W-PhMd41 exhibited a 1.2- to 1.3-fold enhanced activity over their free forms, and the reaction time to reach >99.9 % conversions, with e.e._p of 99.9 % and 98.7 %, was further shortened to 20 h and 7 h, respectively. Additionally, using H1-C61W-PhMd41 as a catalyst, it could achieve a 78.6 % conversion toward (S)-3a after the eighth cycle. This study explored a biosynthesis route for chiral benzo-γ-lactam by using ω-TABm. The immobilization platform constructed with protein crystal as a scaffold is simple, and green, enabling the potential for sustainable application of ω-TABm.