Directed evolution of a serine protease LsPLAase in Bacillus subtilis for high-yield secretory production and efficient depolymerization of polylactic acid

The bio-recyclability of polylactic acid (PLA) is hampered by deficient capacity and low yields of the depolymerases. Whether native production in original isolate Laceyella sacchari LP175 or preparation using Escherichia coli system, the poly( l -lactide) (PLLA) depolymerase, Ls PLAase was restricted for industrial applications by complex downstream purification and limited protein yields. Herein, based on secretory expression system in Bacillus subtilis , purification-free Ls PLAase without solubility tags was obtained, following fermentation optimization, directed evolution, combinatorial mutation, and high-density fermentation, a superior variant, Ls PLAase IWFI (A13I/S136W/S138F/W141I) with boosted performance, achieving protein yield up to 3.21 g/L and enzymatic activity of 6.7 U/mL in 5-L bioreactors, with significant improvement relative to the wild-type (0.9 g/L, 2.4 U/mL) and E. coli system (0.01 g/L, 0.18 U/mL). The prominent variant outperforming ProteinK with superior depolymerization performance toward diverse PLA materials, represented 70% increase in conversion efficiency compared to the wild-type. In all, this study providing a scalable solution for industrial PLA waste recycling to enable circularity.