EndoglucanaseII (Cel5A) of Trichoderma reesei is widely used industrially with the high catalytic efficiency, but it is not stable high temperatures. Structural comparison with the closest thermophilic endoglucanase homolog, Cel5A from Thermoascus aurantiacus, demonstrates disulfide bond differences. Replacement of Cysteine99 with Valine and Cysteine323 with Histidine by site directed mutagenesis caused elimination of two disulfide bonds. Recombinant expression in Pichia pastoris showed the catalytic efficiency (kcat/Km) increment toward CMC for single mutant enzymes, C99V and C323H, about 1.87 and 1.3 folded respectively. This indicates that the elimination of disulfide bond in substrate binding cleft around the catalytic domain of mutant EndoglucanaseII may be increased the flexibility of protein, to form a suitable E-S complex. In direct contrast with previous studies suggesting the existence of disulfide bonds increase the protein stability, the results showed mutant endoglucanase enzymes with disulfide bond reduction have higher thermal stability. The thermal stability of C99V and C323H in 80 °C were increased 2.4 and 2.34 folded, respectively. In this project, theoretical data had a good agreement with the experimental results. Because of high enzyme activity and thermal stability, both of C99V and C323H mutant have high potential suitable for different industrial applications.
Keywords: Disulfide bond; Endoglucanase II; Site directed mutagenesis; Thermal stability.
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