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.

Engineering of a fungal laccase to develop a robust, versatile and highly-expressed biocatalyst for sustainable chemistry

“…51 Although the above laccases hold elongated C-terminal tails that lie closer to the entrance of TNC than in our case, mutations added to PK2 C-terminal tail make it more flexible and may entail a displacement that could improve the access to the TNC channel. 52,53 This hypothesis will be verify when the RY2 variant is crystallized, taking advantage of the high production yields obtained in yeast.…”

Two Decades of Laccases: Advancing Sustainability in the Chemical Industry

“…Structures of some fungal laccase isozymes exhibit an extended C‐terminal, which may block this channel, significantly impairing the catalytic efficiency of the enzyme . A recent study by Hu and coworkers showed that the activity of a laccase from the fungus Pleurotus florida could be increased substantially when its C‐terminal was truncated by 13 residues . Substrate accessibility to the active site also affects catalytic efficiency of the enzyme.…”

Effect of Direct-Current Electric Field on Enzymatic Activity and the Concentration of Laccase

“…At room temperature, a certain molar concentration of laccase was added in buffers with different pH (pH 2.0-6.0, 0.1 mol/l HAc-NaAc) to measure the optimum pH for enzyme activity [9].…”