Electrochemical characterization of a unique, "neutral" laccase from Flammulina velutipes.
The flac1 gene consisted of 1488 bases encodes a novel laccase (Flac1) from Flammulina velutipes. The deduced amino acid sequence of Flac1 with 496 amino acids shows 58-64% homologies with other fungal laccases. The recombinant Flac1 (rFlac1) was heterologously expressed in Pichia pastoris, with sugars of approximately 4 kDa attached on the protein molecule, which has the calculated molecular mass of 53,532 Da. rFlac1 was shown to be a multi-copper oxidase from spectroscopies. The optimum pHs of rFlac1 for oxidations of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and o-aminophenol, were 5.0, 5.0, and 6.0-6.5, respectively, showing higher pH values than those from many other fungal laccases. The slightly acidic or neutral optimum pH that is not strongly dependent on substrates is a unique property of rFlac1. Effective O(2) reduction was realized by the direct electron transfer of rFlac1 at a highly oriented pyrolytic graphite electrode modified with fine carbon particles (Ketjen Black) in O(2)-saturated solution. The pHs showing the maximum ΔE°' [=E°'(enzyme) - E°'(substrate)] coincided well with the optimum pHs shown by rFlac1 under steady-state conditions. The present electrochemical results of rFlac1 indicate that ΔE°' is one of the primary factors to determine the activity of multi-copper oxidases.
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Kaori
K
Otsuka Saito
Shinji
S
Kurose
Yoshio
Y
Tsujino
Toshiyuki
T
Osakai
Kunishige
K
Kataoka
Takeshi
T
Sakurai
Eiichi
E
Tamiya
D000595:Amino Acid Sequence; D000627:Aminophenols / Q000378:metabolism; D001483:Base Sequence; D052160:Benzothiazoles / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D004579:Electron Transport; D055393:Flammulina / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010655:Phenylenediamines / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism
D000627:Aminophenols; D052160:Benzothiazoles; D010655:Phenylenediamines; D011994:Recombinant Proteins; D013451:Sulfonic Acids; C027667:2-aminophenol; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D010088:Oxidoreductases; D042845:Laccase; C024748:copper oxidase; C029728:4-phenylenediamine