Screening and efficient production of engineered lipase B from Candida Antarctica for eco-friendly recycling of waste polycaprolactone

Waste plastics cause serious environmental pollution. Plastic recycling is crucial to combat this global problem. Enzymatic plastic depolymerization is a promising approach for plastic recycling owing to its environmentally friendly nature. Polycaprolactone (PCL), a widely used biodegradable plastic, has been constantly dumped into land and sea. In this study, we screened and produced a PCL-depolymerizing enzyme with high catalytic activity for PCL recycling. Lipase B from Candida Antarctica (CalB) is capable of depolymerizing PCL. Among the three CalB variants with high hydrolytic or synthetic activity reported previously by our group, we found that CalB-658 (V221K and A281I mutations) exhibited higher PCL-depolymerizing activity than the wild-type CalB using a PCL emulsion agar plate. We produced this CalB variant using Pichia pastoris as the recombinant secretory expression host. During the fed-batch fermentation of the recombinant P. pastoris using a 5 L bioreactor, CalB-658 was produced extracellularly at a concentration of 844.3 mg/L with a lipase activity of 13,753.3 U/L. The PCL-depolymerizing activity of CalB-658 was compared with that of the wild-type CalB. During the depolymerization of 60 mg PCL films for 32 h, the wild-type CalB and CalB-658 degraded 54.2% and 97.3% of the PCL film, respectively. Moreover, CalB-658 produced 5.0-fold more 6‑hydroxy hexanoic acid (65 g/L), the monomer of PCL, compared to the wild-type CalB (13 g/L), during a 40 h depolymerization reaction of PCL powder, indicating that CalB-658 exhibited higher PCL-depolymerizing activity compared to the wild-type CalB. This result was confirmed by scanning electron microscopy. Therefore, we have successfully developed a screening and extracellular production system for a PCL-depolymerizing enzyme with high catalytic activity that can produce large quantities of the enzyme with a relatively simple purification step.