Effects of drying methods and additives on the structure, function, and storage stability of subtilisin: role of protein conformation and molecular mobility

Proteins and enzymes for commercial use are often dried in order to extend their shelf lives. This study examined the effects of disaccharides (sucrose and trehalose), polymers (dextran and maltodextrin) and disaccharide–polymer mixtures on the stability of subtilisin, a common industrial laundry detergent enzyme, during drying and during subsequent storage in dried solids containing perborate, a laundry detergent additive that hydrolyzes to form hydrogen peroxide in the presence of water. Subtilisin is susceptible to oxidation by hydrogen peroxide at a partially buried methionine residue, which results in loss of catalytic activity. Two drying methods were compared: spray coating and lyophilization. During both drying methods, the presence of a disaccharide, which can hydrogen bond to subtilisin in the place of lost water, was necessary to inhibit dehydration-induced unfolding of subtilisin. Dehydration-induced unfolding was assessed by second-derivative IR spectroscopy. However, even in the samples in which the protein’s secondary structure was perturbed, the protein refolded to the native conformation and exhibited full activity upon rehydration. None of the additives protected Met222 from oxidation during storage of the initially dry protein formulations at 37 °C and 20 or 75% relative humidity under oxidizing conditions. In contrast, the two additional methionine residues in the enzyme, Met119 and Met175, which are deeply buried in the interior of the protein, remained unmodified during storage of the dried formulations and became oxidized only if the enzyme was completely unfolded in aqueous solution. [Copyright &y& Elsevier]