Enhancement of the thermo-alkali-stability of xylanase II from Aspergillus usamii E001 by site-directed mutagenesis

Abstract

Replacing several serine and threonine residues on the Ser/Thr surface of xylanase II (Xyn II) from Aspergillus usamii E001 with arginines effectively increased the thermostability of the enzyme. The substitution of Ser and Thr residues on the Ser/Thr surface of the enzyme with four (ST4) or five arginines (ST5) led to an increase in optimal temperature of the enzymes by 2 and 5°C for the ST4 and ST5, respectively. The modified enzymes ST4 and ST5 showed 75 and 87% of maximal activities after incubated for 15 min at 55 ℃ compared to only 31% activity for wild-type enzyme. After incubated for 1 h at 55°C, ST4 and ST5 showed 61 and 77% of maximal activity compared to only 27% activity for wild- type enzyme. In addition, these mutations shifted the pH-dependence activity profile to the alkaline region by 1.0 pH units. Kinetic parameters of the four-arginine-substitution enzyme were maintained at the level of the wild-type enzyme with the Km and Vmax values of 5.39±0.20 mg ml-1 and 1410±67 U mg-1 protein, respectively. The five-arginine-substitution enzyme showed only slight alteration in Km and Vmax with Km of 7.68±1.3 mg ml-1 and Vmax of 1161±75 U mg-1 protein, indicating lower substrate affinity and catalytic rate. The study demonstrate that properly introduced arginine residues on the Ser/Thr surface of xylanase might be very effective in the improvement of enzyme thermostability and alkalistability.