Sriprang Rutchadaporn, Asano Krisana, Gobsuk Jarupan, Tanapongpipat Sutipa, Champreda Verawat, Eurwilaichitr Lily
National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Patumthani 12120, Thailand.
J Biotechnol. 2006 Dec 1;126(4):454-62. doi: 10.1016/j.jbiotec.2006.04.031. Epub 2006 May 4.
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.
用四个和五个精氨酸取代黑曲霉BCC14405木聚糖酶Ser/Thr表面的几个丝氨酸和苏氨酸残基，可有效提高该酶的热稳定性。与野生型酶仅15%的活性相比，修饰后的酶在50℃的木聚糖底物中孵育2小时后显示出80%的最大活性。四个精氨酸突变体和五个精氨酸突变体的突变酶半衰期分别增加到257±16分钟和285±10分钟，而野生型酶的半衰期为14±1分钟。因此，精氨酸取代有效地将稳定性提高了18至20倍。四个精氨酸取代酶的动力学参数维持在野生型酶的水平，K(m)和V(max)值分别为8.3±0.1mgml(-1)和9556±66(n = 3)Umg(-1)蛋白质。五个精氨酸取代酶的K(m)和V(max)仅略有变化，K(m)为11.7±1.7mgml(-1)，V(max)为8502±65Umg(-1)蛋白质，表明底物亲和力和催化速率较低。我们的研究表明，在11家族木聚糖酶的Ser/Thr表面适当引入精氨酸残基可能对提高酶的热稳定性非常有效。
