Protein engineering of transaminase facilitating enzyme cascade reaction for the biosynthesis of azasugars.

ABSTRACT

Azasugars, such as 1-deoxynojirimycin (1-DNJ), exhibit unique physiological functions and hold promising applications in medicine and health fields. However, the biosynthesis of 1-DNJ is hindered by the low activity and thermostability of the transaminase. In this study, the transaminase from Mycobacterium vanbaalenii (MvTA) with activity toward d-fructose was engineered through semi-rational design and high-throughput screening method. The final mutant M9-1 demonstrated a remarkable 31.2-fold increase in specific activity and an impressive 200-fold improvement in thermostability compared to the wild-type enzyme. Molecular dynamics (MD) simulations revealed that the mutation sites of H69R and K145R in M9-1 played crucial roles in the binding of the amino acceptor and donor, leading to the stable conformation of substrates within the active pocket. An enzyme cascade reaction was developed using M9-1 and the dehydrogenase from Paenibacillus polymyxa (GutB1) for the production of mannojirimycin (MJ), which provided a new idea for the in vitro biosynthesis of 1-DNJ.