High coenzyme affinity chimeric amine dehydrogenase based on domain engineering

Abstract NADH-dependent phenylalanine amine dehydrogenase (F-AmDH) engineered from (PheDH) catalyzes the synthesis of aromatic chiral amines prochiral ketone substrates. However, its low coenzyme affinity and catalytic efficiency limit industrial application. Here, we developed a chimeric dehydrogenase, cFLF-AmDH, based on relative independence structure at domain level, combined with substrate-binding F-AmDH high-affinity cofactor-binding leucine (L-AmDH). The kinetic parameters indicated that cFLF-AmDH showed twofold improvement in for NADH 4.4-fold increase ( k cat / K m ) compared parent F-AmDH. Meanwhile, also higher thermal stability, half-life increased by 60% 55 °C broader substrate spectrum, than Molecular dynamics simulations suggested constructed had more stable F-AmDH, thereby improving coenzyme. reaction rate 150% reductive amination catalyzed cFLF-AmDH. When NAD + concentration was 0.05 mM, conversion 150%. These results suggest protein shuffling different donors not only cofactor efficiency, but changed specificity stability. Our study highlights engineering is another effective method creating biodiversity properties. Graphical