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Applied Microbiology and Biotechnology

, Volume 60, Issue 6, pp 700–707 | Cite as

Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2

  •  Y. Xiao
  •  X. Tu
  •  J. Wang
  •  M. Zhang
  •  Q. Cheng
  •  W. Zeng
  •  Y. Shi
Original Paper

Abstract.

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 58,522 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°C, respectively. The half-life of the enzyme at 75°C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The Km 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 µM, respectively, and the corresponding Vmax values are 670, 66.8, and 79 µM min–1 mg–1, respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN3 or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 µmol 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 µmol 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.

Keywords

Fermentation Lignin Cyanide MALDI Aromatic Compound 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  •  Y. Xiao
    • 1
  •  X. Tu
    • 1
  •  J. Wang
    • 2
  •  M. Zhang
    • 2
  •  Q. Cheng
    • 2
  •  W. Zeng
    • 1
  •  Y. Shi
    • 1
  1. 1.Key Laboratory of Structural Biology, University of Science and Technology of China, Chinese Academy of Sciences, Hefei, Anhui, 230026, PR China
  2. 2.School of Life Sciences, Anhui University, Hefei, Anhui, 230039, PR China

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