title: Essential role of the N- and C-terminals of laccase from Pleurotus florida on the laccase activity and stability.
abstract: 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△13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1cΔ13-R5V, combined the R5V and △13 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 °C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1cΔ13-R5V. POXA1cΔ13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties.
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Meirong
authors.initials: M
authors.lastname: Hu
authors.forename: Xue
authors.initials: X
authors.lastname: Zhou
authors.forename: Yiping
authors.initials: Y
authors.lastname: Shi
authors.forename: Jianhui
authors.initials: J
authors.lastname: Lin
authors.forename: Muhammad
authors.initials: M
authors.lastname: Irfan
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
mesh_terms: D002244:Carbon / Q000737:chemistry*; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D042845:Laccase / Q000737:chemistry* / Q000302:isolation & purification*; D009584:Nitrogen / Q000737:chemistry*; D020076:Pleurotus / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D002244:Carbon; D042845:Laccase; D009584:Nitrogen