title: Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris.
abstract: Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides, polycyclic aromatic hydrocarbons as well as a very wide range of other compounds with high redox potential. Based on the bias of genetic codons between fungus and yeast, we synthesized a laccase gene GlLCCI, originated from Ganoderma lucidum using optimized codons and a PCR-based two-step DNA synthesis method. The recombinant laccase, GlLCCI was successfully over-expressed in yeast, Pichia pastoris, with an alcohol oxidase1 promoter. The recombinant GlLCCI has a molecular mass of approximately 58 kDa. The K                         (m) values of GlLCCI for 2-2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122 mM, respectively. The V                         (max) of GlLCCI for both substrates was 3,024 and 82.13 μM mg(-1 )min(-1). When ABTS was used as a substrate, the enzyme had an optimal temperature of approximately 55°C. The enzyme was detected over pH values from 2 to 8. The enzyme was strongly activated by K(+), Na(+), Cu(2+) and mannitol. Six amino acids (alanine, histidine, glycine, arginine, aspartate and phenylalanine) increased the catalytic ability of the enzyme. The activity of laccase was obviously inhibited by Fe(2+), Fe(3+), sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62 mg l(-1) of azo dye methyl orange (MO) in cultural medium. With a high MO degradation ability, GlLCCI may have potential in the treatment of industrial effluent containing azo dye MO.
authors.affiliation: College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Tian
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.forename: Hu
authors.initials: H
authors.lastname: Xu
authors.forename: Da-Tong
authors.initials: DT
authors.lastname: Liu
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Chen
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
mesh_terms: D001391:Azo Compounds / Q000378:metabolism; D001673:Biodegradation, Environmental / Q000187:drug effects; D003062:Codon / Q000235:genetics; D003116:Color; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics; D005820:Genetic Testing; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007287:Inorganic Chemicals / Q000494:pharmacology; D007477:Ions; D007700:Kinetics; D042845:Laccase / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D009930:Organic Chemicals / Q000494:pharmacology; D010843:Pichia / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020940:Reishi / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D012995:Solubility / Q000187:drug effects; D013696:Temperature; D013997:Time Factors; D014170:Transformation, Genetic / Q000187:drug effects
chemical_list: D001391:Azo Compounds; D003062:Codon; D007287:Inorganic Chemicals; D007477:Ions; D008670:Metals; D009930:Organic Chemicals; D011994:Recombinant Proteins; C100258:methyl orange; D042845:Laccase