A novel white laccase from Pleurotus ostreatus.
Two laccase isoenzymes (POXA1 and POXA2) produced by Pleurotus ostreatus were purified and fully characterized. POXA1 and POXA2 are monomeric glycoproteins with 3 and 9% carbohydrate content, molecular masses of about 61 and 67 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, of about 54 and 59 kDa by gel filtration in native conditions, and of 61 kDa by matrix-assisted laser desorption ionization mass spectrometry (only for POXA1) and pI values of 6.7 and 4.0, respectively. The N terminus and three tryptic peptides of POXA1 have been sequenced, revealing clear homology with laccases from other microorganisms, whereas POXA2 showed a blocked N terminus. The stability of POXA2 as a function of temperature was particularly low, whereas POXA1 showed remarkable high stability with respect to both pH and temperature. Both enzymes oxidize syringaldazine and ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) together with a variety of different substituted phenols and aromatic amines with the concomitant reduction of oxygen, but POXA1 is unable to oxidize guaiacol. Both enzymes were strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. UV/visible absorption spectra, atomic adsorption, and polarographic data indicated the presence of 4 copper atoms/mol of POXA2 but only one copper, two zinc, and one iron atoms were found/mol of POXA1. The neutral pI and the anomalous metal content of POXA1 laccase render this enzyme unique in its structural characteristics. The lack of typical absorbance at 600 nm allows its classification as a "white" laccase.
Palmieri
G
G
Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, via Mezzocannone 16, I-80134 Naples, Italy.
Giardina
P
P
Bianco
C
C
Scaloni
A
A
Capasso
A
A
Sannia
G
G
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology*; D002384:Catalysis; D003300:Copper / Q000032:analysis; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000032:analysis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D042845:Laccase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D015032:Zinc / Q000032:analysis
D005656:Fungal Proteins; D007527:Isoenzymes; D003300:Copper; D007501:Iron; D010088:Oxidoreductases; D042845:Laccase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D015032:Zinc