An in-silico and experimental dual-screening strategy for robust lyoprotection of restriction endonucleases

An in-silico and experimental dual-screening lyophilization strategy was developed for long-term ambient storage of two model restriction endonucleases (REase) of XbaI (oxidation-resistant) and XhoI (oxidation-sensitive). After molecular docking and experimental screening of lyoprotectants and their combinations, a trehalose-povidone-arginine ternary lyoprotectant "combo" was finally developed. The lyophilized formulations were primarily evaluated by four critical quality attributes (CQAs), including lyocake appearance, reconstitution rate, relative enzymatic activity (REA) and short-term storage stability. Four ternary lyophilized formulation candidates were further evaluated by thermogravimetric analysis (TGA) for the residual moisture contents (<2%). Based on the results of four CQAs and TGA, the optimized formulation was finally determined, including trehalose (15%), arginine (5%) and povidone (5%). Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) results indicated that REase lyocakes formed a co-amorphous glassy matrix. Scanning electron microscopy (SEM) images showed REase lyocakes with intact, loose and porous structure. Following six months of long-term stability testing under controlled conditions (25 °C, 60% RH), the lyophilized REase products still maintained comparable DNA cleavage efficiency to that of freshly prepared samples. This study presents a unique in-silico and experimental dual-screening strategy for developing cold-chain-free solid-state enzyme reagents.