Direct evolution and expression of Tfcut-B3 in Bacillus subtilis for efficient depolymerization of commercial...

Enzymatic depolymerization is a potentially efficient and environmentally friendly solution for poly(butylene adipate-co-terephthalate) (PBAT) waste recycling and utilization. However, the depolymerization capacity of reported depolymerases against diverse, real-world commercial PBAT films remains unclear, hindering accurate evaluation of their utility. In this study, a mutant PBAT depolymerase (Tfcut-B3) with enhanced thermal stability and improved activity was obtained through directed evolution of Tfcut-DM. Tfcut-B3 completely depolymerized PBAT white film into terephthalic acid (TPA) within 48 h, yielding 2.0 and 1.3 times more TPA than Tfcut-DM and Tfcut-DM(Q132Y), respectively. Tfcut-B3 completely degraded 8/10 commercial PBAT-based films into TPA within 48 h, with the remaining two (PB-6 and PM-4) being broken down into small fragments; Tfcut-DM was unable to completely degrade any of the films. Tfcut-B3 overexpression and extracellular secretion in Bacillus subtilis was achieved using chromosomal integration; in flask fermentation, a Tfcut-B3 expression level of 274 mg/L was achieved through promoter and host optimization. Tfcut-B3 fermentation supernatant completely degraded up to 64.9 mg of PBAT white film within a 20 mL system at 72 h. These findings provide a theoretical basis for the large-scale application of PBAT depolymerases.