Extended Data Fig. 4: Multilevel screening approach followed for PAM variant selection.
From: An engineered enzyme embedded into PLA to make self-biodegradable plastic
Structure-based engineering of PAM PLAase consisted in four levels of screening of PAM mono-variants before recombinational analysis and the evaluation of performances of two triple variants (PAMFLI and PAMFFI). The 1st level of screening evaluates the ability to form a halo on dispersed PLA submicroparticles at pH 9 and 45 °C of 133 PAM mono-variants resulting from the site-saturation mutagenesis of seven selected amino acid positions. The 2nd level of screening, through liquid PLA depolymerization, evaluates PLA depolymerization specific activity of selected variants at pH 9 and 45 °C. The best variants identified entered in the 3rd level of screening that evaluates PLA depolymerization performances at pH 7.5 and 45 °C. Thermostability assessment performed during the 4th level of screening finalizes the selection of the most promising mono-variants of PAM.
