Synergistic Mutations Create Bacillus Subtilisin Variants with Enhanced Poly-L-Lactic Acid Depolymerization Activity

Enzymatic recycling of poly -L-lactic acid (PLLA) plastic has recently become an area of interest; however, investigation of enzymatic mechanisms and engineering strategies to improve activity remains limited. In this study, we have identified a subtilisin from Bacillus pumilus that has the ability to depolymerize high-molecular-weight PLLA. We performed a comparative, muta-tional analysis of this enzyme with a less active homologue from Bacillus subtilis to determine residues favored for activity. Our results demonstrate that both enzymes contain residues favored for PLLA depolymerization, with the generation of several hyperactive variants. In silico modeling suggests that increases in activity are due to opening of the binding pockets and increased surface hydrophobicity. Combinations of hyperactive mutations have synergistic effects with the generation of subtilisin variants with 830-and 184-fold increases in activity for B. subtilis and B. pumilus subtilisins, respectively. One B. pumilus subtilisin variant can visibly dissolve high-molecular-weight PLLA films.

Automated summary

A subtilisin enzyme from Bacillus pumilus has been identified to have the ability to depolymerize high-molecular-weight PLLA. Mutational analysis of this enzyme and comparison with a less active homologue from Bacillus subtilis has revealed residues favored for PLLA depolymerization. Hyperactive variants of the enzyme have been generated, with increased activity attributed to opening of binding pockets and increased hydrophobicity. Synergistic effects are observed with combinations of hyperactive mutations, resulting in 830- and 184-fold increases in activity for B. subtilis and B. pumilus subtilisins, respectively. One variant of B. pumilus subtilisin can visibly dissolve high-molecular-weight PLLA films.