Improvement of thermostability of fungal xylanase by using site-directed mutagenesis

Replacing several serine and threonine residues on the Ser/Thr surface of the xylanase from Aspergillus niger BCC14405 with four and five arginines effectively increases the thermostability of the enzyme. The modified enzymes showed 80% of maximal activity after incubating in xylan substrate for 2h at 50 degrees C compared to only 15% activity for wild-type enzyme. The half-life of the mutated enzymes increased to 257 +/- 16 and 285 +/- 10 min for the four- and five-arginine mutants, respectively, compared to 14 +/- 1 min for the wild-type enzyme. Thus, the arginine substitutions effectively increase stability by 18-20-fold. Kinetic parameters of the four-arginine-substitution enzyme were maintained at the level of the wild-type enzyme with the K-m and V-max values of 8.3 +/- 0.1 mg ml(-1) and 9556 +/- 66 (n = 3) U mg(-1) protein, respectively. The five-arginine-substitution enzyme showed only slight alteration in K-m and V-max with K-m of 11.7 +/- 1.7 mg ml(-1) and V-max of 8502 +/- 65 U mg(-1) protein, indicating lower substrate affinity and catalytic rate. Our study demonstrated that properly introduced arginine residues on the Ser/Thr surface of xylanase family 11 might be very effective in improvement of enzyme thermostability. (c) 2006 Elsevier B.V. All rights reserved.