A laccase with antiproliferative activity against tumor cells from an edible mushroom, white common Agrocybe cylindracea
Introduction
Proteins with antiproliferative/antitumor (Wang et al., 1995, Wang et al., 1996), antifungal (Lam and Ng, 2001, Wang and Ng, 2004a) and HIV-1 reverse transcriptase inhibitory (Lam and Ng, 2001, Wang and Ng, 2001a) activities have been purified and characterized from mushrooms including laccases (Wang and Ng 2004b), antifungal proteins (Lam and Ng, 2001, Wang and Ng, 2004c), ribonucleases, ribosome-inactivating proteins (Wang and Ng, 2000a, Wang and Ng, 2001b), ubiquitin-like peptides (Wang et al. 2003) and lectins (Wang et al., 1995, Wang et al., 1996, Wang et al., 1998, Yagi et al., 1997, Wang and Ng, 2004d).
From the mushroom Agrocybe cylindracea, an antifungal protein (Ngai et al. 2005), a ubiquitin-like protein (Ngai et al. 2003), a lectin (Liu et al. 2008), a haloperoxidase (Ullrich and Hofrichter 2005), a phytase (Lassen et al. 2001), a hemolysin (Ngai and Ng 2006), a polysaccharide (Yoshida et al. 1996), antimutagenic factors (Taira et al. 2005), cyclooxygenase inhibitory and antioxidant compounds (Zhang et al. 2003), have been isolated. The activities of laccase at different developmental stages of A. cylindracea have been determined (Fu et al. 2006). However, no laccase has been purified.
Laccases are lignin-degrading enzymes (Evans et al. 1994) that are used in textile dyes, pulping, biosensors, detoxification of polluted water and other biotechnological processes (Brenna and Bianchi, 1994, Palmieri et al., 1994, Reid and Paice, 1994, Ghindilis et al., 1995, Martirani et al., 1996). In view of the importance of laccases, the present study aimed to isolate a laccase from A. cylindracea. The results would add to the existing knowledge about this mushroom especially its proteinaceous constituents since only about a handful of proteins have been reported to date. Hitherto laccases have been isolated from a number of mushrooms (Eggert et al., 1996, Munoz et al., 1997, Cambria et al., 2000, Dedeyan et al., 2000, Shin and Lee, 2000, Garzillo et al., 2001, Rogalski et al., 2001, Murugesan et al., 2006, Wang and Ng, 2006a, Revankar et al., 2007), mostly from their mycelia. Only some of the laccases are from fruiting bodies e.g. Pleurotus eryngii (Wang and Ng 2006b) and Hericium erinaceum (Wang and Ng 2004e). The present investigation disclosed that the fruiting bodies of the mushroom A. cylindracea produce a laccase with HIV-1 reverse transcriptase inhibitory activity and antiproliferative activity against HepG2 cells and MCF7 cells.
Section snippets
Isolation of laccase
Fresh fruiting bodies (1 kg) of the mushroom A. cylindracea were homogenized in distilled water (3 ml/g) using a blender. The homogenate was centrifuged (14,000 × g, 25 min, 4 °C), and the supernatant was collected. Tris–HCl buffer (1 M, pH 7.4) was added to the supernatant until the concentration of Tris reached 10 mM. The supernatant was then subjected to chromatography on a column (5 cm × 20 cm) of DEAE-cellulose (Sigma) in 10 mM Tris–HCl buffer (pH 7.4). Unadsorbed proteins (fraction D1) were eluted
Results
Ion exchange chromatography of the fruiting body extract on DEAE-cellulose produced an unadsorbed fraction D1 and two adsorbed fractions D2 and D3. Laccase activity was enriched in fraction D2 (Table 1). Upon chromatography on SP-Sepharose, D2 was resolved into an unadsorbed fraction SP1, and three adsorbed fractions SP2, SP3 and SP4 (Fig. 1). Fraction SP1 with laccase activity (Table 1) was chromatographed on Q-Sepharose to give an unadsorbed fraction Q1, and three adsorbed fractions Q2, Q3
Discussion
In the present study, a laccase was successfully isolated from the fruiting bodies of the edible mushroom A. cylindracea. Ion exchange chromatography on DEAE-cellulose, SP-Sepharose, Q-Sepharose, and gel filtration on Superdex 75 were used successively to remove inactive proteins from the laccase-containing chromatographic fraction. Each of the aforementioned chromatographic media was effective in yielding a laccase-enriched fraction separable from a fraction with little or no laccase activity.
Acknowledgments
This work was financially supported by National Grants of China (2006BAD07A01 and nyhyzx07-008) and a direct grant from the Medicine Panel, CUHK Research Committee, was gratefully acknowledged.
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