Trehalose decorated nanostructures stabilize combined cross-linked enzyme aggregates (Combi-CLEAs) of β-galactosidase and glucose isomerase.

Combined cross-linked enzyme aggregates (Combi-CLEAs) of β-Galactosidase (β-Gal) and Glucose Isomerase (GI) allow the transformation of d-lactose to lactose-fructose syrup through one-pot cascade biocatalytic reactions. Despite its promise, the low thermostability of β-Gal and high-temperature demands for GI limits this application. Trehalose is a protein-stabilizing disaccharide which has been utilized in immobilized enzyme systems to enhance protein thermostability. In this work, trehalose decorated poly (amidoamine) (PAMAM) dendrimers were synthesized at low and high surface coverage levels (10 % and 50 %) and used to stabilize Combi-CLEAs of β-Gal and GI. Addition of trehalose decorated nanostructures to β-Gal and GI Combi-CLEAs enhanced β-Gal performance at elevated temperatures (58.6 % higher activity and 2.26× higher stability) while maintaining GI performance of Combi-CLEAs. The resulting Combi-CLEAs containing trehalose decorated nanostructures retained ~40 % β-Gal activity following 7 consecutive reaction cycles and exhibited GI activity for 5 consecutive reaction cycles. Overall, this study demonstrates the potential of trehalose decorated nanostructures to stabilize proteins at elevated temperatures typical of bioprocessing applications and storage of therapeutics.