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 (CH2)(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 mu mol/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 front 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. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 111-117, 2010