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 mgml(-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 mgml(-1) and V(max) of 8502+/-65 Umg(-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.
Sriprang
Rutchadaporn
R
National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Patumthani 12120, Thailand. rutchadaporn@biotec.or.th
Asano
Krisana
K
Gobsuk
Jarupan
J
Tanapongpipat
Sutipa
S
Champreda
Verawat
V
Eurwilaichitr
Lily
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D001120:Arginine; D043364:Endo-1,4-beta Xylanases