Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro.
In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144 mgl(-1) laccase in yeast, together with a 1.4-fold increase in K(cat). Thus, the total activity is enhanced 5-fold for 2,2'-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6mM copper, the highest activity value reached 30 Uml(-1) after a 3-day cultivation at a temperature of 30 degrees C(.) In comparison with the wild type, the best mutant enzymatic properties (K(m) for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46 A. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.
Hu
Mei-Rong
MR
State Key Laboratory of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Chao
Ya-Peng
YP
Zhang
Guo-Qing
GQ
Yang
Xiu-Qing
XQ
Xue
Zhi-Quan
ZQ
Qian
Shi-Jun
SJ
D052160:Benzothiazoles / Q000737:chemistry; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D005020:Ethyl Methanesulfonate / Q000737:chemistry*; D042845:Laccase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis / Q000187:drug effects* / Q000235:genetics; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000737:chemistry; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics
D052160:Benzothiazoles; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D005020:Ethyl Methanesulfonate; D042845:Laccase