Expression and characterization of Ganoderma fornicatum laccase in Pichia pastoris and the role of glycosylation for laccase stability
Date Issued
2010
Date
2010
Author(s)
Huang, Wan-Ting
Abstract
Laccases, a group of copper-containing oxidizing enzymes, catalyze the oxidation of various aromatic compounds, particularly phenolic substrates with the concomitant reduction of molecular oxygen to water. This characteristic makes laccases of great potential for industrial applications, including bioremediation, lignocelluloses processings, organic syntheses, etc. White-rot fungi have been deemed as a good resource of laccase genes due to their high ligninolytic activity. In this study, the newly defined laccase gene, lac1, from the medicinal white-rot fungus Ganoderma fornicatum 0814 was cloned and expressed in Pichia pastoris. The corresponding open reading frame has 1563 nucleotides and encodes a protein of 521 amino acids, including secretion signal with a 21-residue peptide. The ten amino acids downstream of a conserved Cys residue existing within the laccases encoded by the lac1 are phenylalanine, suggesting that the laccases from G. fornicatum 0814 may demonstrate high redox potential and good ligninolytic activity. The absorption spectrum of the purified laccase rLac1 indicated that it is a yellow laccase rather than the common blue laccase. The optimum pH of rLac1 was 2.5, 3.0 and 3.0 for ABTS, DMP and guaiacol, respectively. The kinetic parameter Km of rLac1 was 103.9, 276.7 and 1263.4 uM for ABTS, DMP and guaiacol, respectively. The optimal temperature for rLac1 was 55°C for all of the substrates. rLac1 is stable up to 60°C for 3 h and in the pH range 2.5 to 10.0 for 24 h. Sodium azide and thioglycolic acid strongly inhibited rLac1 activity, but it was not affected by EDTA. rLac1 retained more than 80% activity in 50% (v/v) ethanol, methanol, DMF and DMSO after incubation at 25°C for three-hour. Less than 20% of activity was found in 50% (v/v) acetone and acetonitrile after incubation at 25°C for three-hour. The results show that rLac1 is thermostable, pH-stable and tolerant to organic solvents. It has also been produced in fermentations of P. pastoris with synthetic media. These properties suggest that rLac1 has potential uses in industrial applications. To enhance laccase expression in P. pastoris, lac1 was modified to the optimal codon usage of P. pastoris. The expression level was thus increased up to 30%. Glycosylation retards protein overexpression, although it helps stabilize enzymes and avoids protein degradation. Through sequence alignment, it is shown that laccase from G. fonicatum has more glycosylation sites than that from other species. Enzyme activity decreases drastically when the asparagine of some glycosylation sites were replaced by aspartic acid (N7D, N168D, N190D, N302D and N334D) by site-directed mutagensis. With the deletion of the glycosylation site N4 (N190D), only 32% of the laccase activity remains. Only 20-27% activity remains if 1-4 more glycosylation site is deleted, in addition to the deletion of the N4 site. Thus, N4 glycosylation site had a critical effect on the activity of the laccase. Moreover, the thermal stability and actonitrile- and ethanol-tolerance were affected when the N7 glycosylation site was deleted (N334D). Any individual site deletion had little effect on the pH-stability. However, multiple site deletiond had significant effection the pH-stability. It was deduced that glycosylation at these specific sites are important for the activity and stability of laccase (Lac1) from G. fonicatum.
Subjects
Ganoderma fornicatum
Pichia pastoris
laccase
glycosylation
Type
thesis
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