A laccase with antiproliferative activity against tumor cells from an edible mushroom, white common Agrocybe cylindracea

Abstract

A laccase, with HIV-1 reverse transcriptase inhibitory activity (IC₅₀ = 12.7 μM) and antiproliferative activity against HepG2 cells (IC₅₀ = 5.6 μM) and MCF7 cells (IC₅₀ = 6.5 μM), was purified from fresh fruiting bodies of the edible white common Agrocybe cylindracea mushroom. The laccase, which had a novel N-terminal sequence, displayed a molecular mass of 58 kDa within the range reported for most other mushroom laccases. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, SP-Sepharose, and Q-Sepharose and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but unadsorbed on SP-Sepharose. Its optimum pH was pH 3–4 and its optimum temperature was 50 °C. The activity of the isolated laccase differed from one substrate to another. The ranking was ABTS > N,N-dimethyl-1,4-phenylenediamine > hydroquinone > catechol > 2-methylcatechol > pyrogallol.

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.