Characterization of a Coprinus cinereus laccase

被引:110
作者
Schneider, P
Caspersen, MB
Mondorf, K
Halkier, T
Skov, LK
Ostergaard, PR
Brown, KM
Brown, SH
Xu, F
机构
[1] Novo Nordisk AS, DK-2880 Bagsvaerd, Denmark
[2] Univ Copenhagen, Dept Chem, DK-2100 Copenhagen, Denmark
[3] Novo Nordisk AS, Biotech, Davis, CA 95616 USA
关键词
laccase; redox potential; electron paramagnetic resonance; activity; stability;
D O I
10.1016/S0141-0229(99)00085-X
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A wild-type Coprinus cinereus laccase and its recombinant form expressed in an Aspergillus oryzae host have been purified and characterized. The mature laccase had a molecular mass of 58 kDa by mass spectrometry, an isoelectric point near 4, and two absorption maxima at 278 and 614 nm. Photometric titration with 2,2'-biquinoline showed a Cu/protein(subunit) stoichiometry of approximate to 4. The electron paramagnetic resonance spectrum showed typical type 1 and type 2 Cu signals, and the circular dichroism showed a typical coordination geometry of the type 1 Cu(II). At pH 5.5, the enzyme had a redox potential of 0.55 V vs. normal hydrogen electrode at its type 1 site. The laccase could oxidize 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) and syringaldazine with optimum pH of 4 and 6.5, respectively. Halides inhibited the laccase. At pH 8.5, the laccase had an optimum temperature between 60 degrees C and 70 degrees C. At the same pH, the laccase had a half-life of >200 or 21.8 min in the presence of 0 or 2 mM H2O2, respectively, at 40 degrees C. Mediated by several phenols and phenothiazines, the laccase was able to oxidatively bleach Direct Blue 1 dye at alkaline pH, making it a promising industrial enzyme candidate. (C) 1999 Elsevier Science Inc. All rights reserved.
引用
收藏
页码:502 / 508
页数:7
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