Abstract
A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase.
Highlights
The mycelium of the mushroom Lentinus tigrinus HPXG59 was incubated at 25◦C in a shaker for 7 days
After the flow through fraction (D1) had been eluted, the column was eluted stepwise with 0.1 M NaCl and 1 M NaCl in the starting buffer to yield fractions D2 and D3
Fraction D3 was subsequently subjected to ion exchange chromatography on a 2.5 × 20 cm column of CM-cellulose (Sigma) in 10 mM NH4OAc when the flow through fraction (CM1) had all been eluted, the column was eluted with a linear concentration (0-1 M) gradient of NaCl in 10 mM NH4OAc buffer
Summary
Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) are glycosylated multicopper oxidases. Laccases are widely distributed among plants [2], insects [3], bacteria [4, 5], and basidiomycetous and ascomycetous fungi [6,7,8]. They have been purified from cultured mycelia of various mushrooms [9,10,11,12,13]. Proteins with HIV-1 reverse transcriptase inhibitory activities have been purified and characterized from mushrooms including laccase [17,18,19], antifungal proteins [20], ribonucleases [21], ubiquitin-like peptides [22], and lectins [23]
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