The introduction of disulfide bonds has been used as a strategy to enhance the stability of Bacillus circulans xylanase. The transition temperature of the S100C/N148C (DS1), V98C/A152C (DS2), and A1GC/G187,C188 (cXl) in comparison to the wild type was increased by 5.0, 4.1 and 3.8 degrees C, respectively. Interestingly, a combination of two disulfide bonds of DS1 and cXl (cDS1, circular disulfide 1) led to a 12 degrees C increase in the transition temperature. Importantly, an increase in the melting point and DeltaDeltaG values of the cDS1 mutant was cooperative. These results suggest that the mechanism of stabilization by disulfide bonds under irreversible denaturation condition is achieved through: (1) a change in the rate-limiting step on the denaturation pathway; (2) destabilizing the unfolded state without affecting the relative rate constants on the denaturation pathway (like cXl mutant); and (3) or combination of the two (cDS1 mutant).