Stabilization of invertase by molecular engineering

Extracellular invertase (EC 3.2.1.26) of Saccharomyces cerevisiae was stabilized against thermal denaturation by intermolecular and intramolecular crosslinking of the surface nucleophilic functional groups with diisocyanate homobifunctional reagents (O==C==N(CH(2))(n)N==C==O) of various lengths (n = 4, 6, 8). Crosslinking with 1,4-diisocyanatobutane (n = 4) proved most effective in enhancing thermostability. Stability was improved dramatically by crosslinking 0.5 mg/mL of protein with 30 mumol/mL of the reagent. Molecular engineering by crosslinking reduced the first-order thermal denaturation constant at 60 degrees C from 1.567 min(-1) (for the native enzyme) to 0.437 min(-1) (for the stabilized enzyme). Similarly, the best crosslinking treatment increased the activation energy for denaturation from 391 kJ mol(-1) (for the native protein) to 466 kJ mol(-1) (for the stabilized enzyme). Crosslinking was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.