Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2.
A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.
Xiao
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YZ
Key Laboratory of Structural Biology, University of Science and Technology of China, Chinese Academy of Sciences, Hefei, Anhui, 230026, PR China.
Tu
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XM
Wang
J
J
Zhang
M
M
Cheng
Q
Q
Zeng
W Y
WY
Shi
Y Y
YY
D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D002847:Chromatography, Agarose; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005800:Genes, Fungal; D006841:Hydrocarbons, Aromatic / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D042845:Laccase; D008031:Lignin / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D012997:Solvents; D013379:Substrate Specificity; D013696:Temperature; D014462:Ultrafiltration
D005656:Fungal Proteins; D006841:Hydrocarbons, Aromatic; D012997:Solvents; D008031:Lignin; D010088:Oxidoreductases; D042845:Laccase