A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the Mushroom Lentinus tigrinus

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.

Lentinus tigrinus is the white rot fungus, which are the most efficient lignin degraders in nature and are capable of producing laccases and ligninolytic peroxidases [13,24].
In view of the observation that laccases from other strains (Strain 8/18, Strain CBS 577.79, and Strain BKM F3616D) exhibit different molecular mass and characteristics [29][30][31][32], and no attention on its medicinal effects, this prompted us to isolate a laccase with inhibitory activity against HIV-1 reverse transcriptase from the mycelium of the L. tigrinus HPXG59 and compare its characteristics with laccases isolated from other L. tigrinus strains reported earlier.

Purification and Analysis of Laccase.
The mycelium of the mushroom Lentinus tigrinus HPXG59 was incubated at 25 • C in a shaker for 7 days. The incubation broth was collected

Analysis of N-Terminal Amino Acid Sequence.
Amino acid sequence analysis was carried out using an HP G1000A Edman degradation unit and an HP1000 HPLC system [20].

Assay of pH and Temperature Optima.
In the assay for optimal pH value, a series of solution of ABTS in buffers with different pH values was used. The assay buffers were prepared in 0.1 M NaOAc buffer (pH 3.0, pH 4.0, and pH 5.0), 0.1 M MES buffer (pH 5.0, pH 6.0, and pH 7.0) and 0.1 M HEPES buffer (pH 7.0, pH 8.0, and pH 9.0). The assay temperature was 37 • C. To determine the optimal temperature, the reaction mixture was incubated at 20 • C to 90 • C.

Assay for HIV-1 Reverse Transcriptase Inhibitory Activity.
The assay for HIV reverse transcriptase inhibitory activity was carried out according to instructions supplied with the assay kit from Boehringer Mannheim (Germany). The assay takes advantage of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly (A) oligo (dT) 15. The digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same DNA molecule, which is freshly synthesized by the reverse transcriptase (RT). The detection and quantification of synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol. Biotin-labeled DNA binds to the surface of microtiter plate modules that have been precoated with streptavidin. In the next step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigenin-labeled DNA. In the final step, the peroxidase substrate is added. The peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples at 405 nm can be determined by using a microtiter plate (ELISA) reader and is directly correlated to the level of RT activity. A fixed amount (4-6 ng) of recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of the isolated laccase was calculated as percent inhibition as compared to a control without the laccase [18].

Purification of Laccase.
The mycelial pellet extract of Lentinus tigrinus was fractionated by ion exchange chromatography on DEAE-cellulose. Laccase activity was undetectable in fraction D1, low fraction D2, and concentrated in fraction D3 (Table 1). D3 was resolved on CM-cellulose into a large unadsorbed fraction CM1, a small adsorbed fraction CM2, and a large adsorbed fraction CM3 (Figure 1). Laccase activity was indiscernible in CM1 and mainly located in CM2. Fraction CM3 had very little activity (Table 1). Gel filtration of CM2 yielded two peaks of roughly equal size (collected as fraction SU1 and SU2) together with a small peak SU3 adjacent to SU2 (Figure 2). Laccase activity was enriched in SU1. There was residual activity in SU2 and no activity in SU3 (Table 1). Peak SU1 represented the purified laccase. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The purified laccase exhibited an activity of 76.6 U/mg ( Table 1). The chromatographic behavior of Lentinus tigrinus laccase on cationic and anionic exchangers was similar to that of Tricholoma mongolicum laccase [35]. They were both adsorbed on DEAE and CM ion exchangers. But it was differed from laccase from Lentinus edodes [36][37][38] and other mushrooms [18,19,42] (Table 3). The purity was determined by SDS-PAGE. This observation suggests that this enzyme is a monomeric protein (Figure 3).

Determination of Molecular
Mass. SU1 appeared as a single band with a molecular mass of 59 kDa in SDS-PAGE ( Figure 3) and as a single peak with the same molecular mass upon re-chromatography on Superdex 75 (data not shown). Its molecular mass was close to those of most laccases.
The laccase from L. tigrinus demonstrated a molecular mass of 59 kDa, which is less than those (63 kDa and 69.1 kDa) of L. tigrinus reported earlier [29,32,33]    masses from 30 to 70 kDa is demonstrated by laccase from mushroom (Table 3).
Journal of Biomedicine and Biotechnology was only a slight similarity to the N-terminal sequences of L. tigrinus reported earlier [32]. It is noteworthy that laccases from different L. tigrinus strains may have distinct N-terminal sequence.

3.4.
Optimal pH and Temperature. The activity increased slightly as the ambient pH was elevated to 4. There was a precipitous decline in activity when the pH was 5. Activity was barely detectable at pH 6 and indiscernible at and above pH 7 (Figure 4). The laccase activity rose steadily as the ambient temperature was elevated from 20 • C to 60 • C. The activity dropped abruptly when the temperature rose to 70 • C. At 100 • C no activity remained ( Figure 5). L. tigrinus laccase requires a temperature of 60 • C to express maximal activity. In this aspect it resembles laccases from Clitocybe maxima and Tricholoma matsutake which have the optimum temperature at 60 • C. A pH of 4 is optimal for the activity of L. tigrinus laccase requires an acetic pH for activity, like laccases from a number of mushrooms including Agaricus blazei.

HIV-1 Reverse Transcriptase Inhibitory
Activity. The laccase inhibited HIV-1 reverse transcriptase by 27.7% and 86.3% when the laccase concentration was 1.5 μM and 15 μM, respectively. The IC 50 value was 2.4 μM. A comparison of IC 50 value towards HIV-1 RT from mushroom laccase is shown in Table 3.
Another 69.1 kDa laccase was isolated from mycelia of P. tigrinus CBS 577.79, by employing ultrafiltration (cutoff 10 kDa), chromatography on Q-Sepharose sn Superdex 75 [29]. Cadimaliev has reported a laccase which is isolated by chromatography on TEAE-cellulose and DEAE-Toyopearl 650 M from P. tigrinus BKM F3616D [30]. However, its molecular mass and N-sequence has not been described. The laccases isolated by Leontievsky and Golovleva differ from the laccase isolated in the present study and the laccase purified by Daniele Quaratino in biological activities, such as in molecular mass, optimum temperature, and optimum pH (Table 3). It is hard to compare the N-sequence, since there is no report about the laccase isolated by Leontievsky, Golovleva, and Cadimaliev.

Conclusions
In summary, the L. tigrinus laccase was found to be different from those of previous laccase in N-terminal sequence, molecular mass, optimal pH, and optimal temperature. The laccase inhibited HIV-1 reverse transcriptase with IC 50 value of 2.4 μM but was devoid of any antifungal or anti-prolif-er-a-tive activity. Previously isolated L. tigrinus laccases have not been so tested [29,30,32]. This study yielded information about the potentially exploitable activities of L. tigrinus laccase.