Essential Role of the N- and C-terminals of Laccase from Pleurotus florida on the Laccase Activity and Stability----中国科学院微生物研究所

科研成果

论文题目:	Essential Role of the N- and C-terminals of Laccase from Pleurotus florida on the Laccase Activity and Stability
作者:	Hu Meirong, Zhou Xue, Shi Yiping, Lin Jianhui, Irfan Muhammad, Tao Yong*
联系作者:	Tao Yong*
刊物名称:	Appl Biochem Biotechnol
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年份:	2014
影响因子:	1.994
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摘要:	POXA1b is the most thermostable laccase isoenzyme from Pleurotus ostreatus. POXA1b is remarkably stable at alkaline pH (the t1/2 at pH 10 was 30 days), and its C-terminal affects its catalytic and stability properties. We cloned POXA1c from P. florida, which showed 99 % identity with POXA1b. POXA1c was functionally expressed in Pichia pastoris. The functions of the N and C termini of POXA1c were investigated using site-directed mutagenesis. Compared with POXA1c, the N-terminal R5V site effectively increased the specific activities for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol by 2- and 3.5-fold, respectively. A C-terminal truncated mutant, POXA1c big up tri, open13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1cDelta13-R5V, combined the R5V and big up tri, open13 effects. The specific activity of this double mutant for ABTS was 1,321 U/mg, which indicated a 4-fold increase compared with the wild type. The role of residue V5 on laccase catalytic properties was also observed for laccases from Trametes versicolor and Rigidoporus lignosus. The specific activities of the V5R of the laccases from T. versicolor and R. lignosus were half of that of the wild type. The pH and thermal stability analysis of POXA1c and its mutants showed that the enzymes were remarkably stable because they showed 63 % residual activity after incubation for 108 h at 30 degrees C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1cDelta13-R5V. POXA1cDelta13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties.