title: Differential Expression of Laccase Genes in Pleurotus ostreatus and Biochemical Characterization of Laccase Isozymes Produced in Pichia pastoris.
abstract: In this study, transcriptome analysis of twelve laccase genes in Pleurotus ostreatus revealed that their expression was differentially regulated at different developmental stages. Lacc5 and Lacc12 were specifically expressed in fruiting bodies and primordia, respectively, whereas Lacc6 was expressed at all developmental stages. Lacc1 and Lacc3 were specific to the mycelial stage in solid medium. In order to investigate their biochemical characteristics, these laccases were heterologously expressed in Pichia pastoris using the pPICHOLI-2 expression vector. Expression of the laccases was facilitated by intermittent addition of methanol as an inducer and sole carbon source, in order to reduce the toxic effects associated with high methanol concentration. The highest expression was observed when the recombinant yeast cells were grown for 5 days at 15℃ with intermittent addition of 1% methanol at a 12-hr interval. Investigation of enzyme kinetics using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate revealed that the primordium-specific laccase Lacc12 was 5.4-fold less active than Lacc6 at low substrate concentration with respect to ABTS oxidation activity. The optimal pH and temperature of Lacc12 were 0.5 pH units and 5℃ higher than those of Lacc6. Lacc12 showed maximal activity at pH 3.5 and 50℃, which may reflect the physiological conditions at the primordiation stage.
authors.affiliation: Division of Applied Life Science and Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea.
authors.forename: Minsa
authors.initials: M
authors.lastname: Park
authors.affiliation: Division of Applied Life Science and Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea.
authors.forename: Minseek
authors.initials: M
authors.lastname: Kim
authors.affiliation: Division of Applied Life Science and Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea.
authors.forename: Sinil
authors.initials: S
authors.lastname: Kim
authors.affiliation: Division of Applied Life Science and Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea.
authors.forename: Byeongsuk
authors.initials: B
authors.lastname: Ha
authors.affiliation: Division of Applied Life Science and Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea.
authors.forename: Hyeon-Su
authors.initials: HS
authors.lastname: Ro
keywords: Heterologous expression; Laccase; Mushroom; Primordia