Purification and characterization of a laccase from the edible wild mushroom Tricholoma mongolicum.
A novel laccase from Tricholoma mongolicum was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The purified enzyme was obtained with a specific activity of 1480 U/mg-protein and a final yield of 15%. It was found to be a monomeric protein with a molecular mass of 66 kDa as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was GIGPVADLYVGNRIL, similar to some but different other mushroom laccase. The optimum pH and temperature for the purified enzyme were pH 2 to pH 3 and 30 degrees C, respectively. It displayed a low K(m) toward 2,7-azinobis (3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) and high K(cat)/K(m) values. The purified laccase oxidized a wide range of lignin-related phenols, but exerted maximal activity on ABTS. It was significantly inhibited by Hg(2+) ions, and remarkably stimulated by Cu(2+) ions. It inhibited HIV-1 reverse transcriptase and proliferation of hepatoma HepG2 cells and breast cancer MCF7 cells with an IC50 of 0.65 microM, 1.4 microM, and 4.2 microM, respectively, indicating that it is also an antipathogenic protein.
Li
Miao
M
State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing.
Zhang
Guoqing
G
Wang
Hexiang
H
Ng
Tzibun
T
D000595:Amino Acid Sequence; D052160:Benzothiazoles / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D013451:Sulfonic Acids / Q000378:metabolism; D055439:Tricholoma / Q000737:chemistry / Q000201:enzymology*
D052160:Benzothiazoles; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D042845:Laccase; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase