Purification and characterization of an alkaline chloride-tolerant laccase from a halotolerant bacterium, Bacillus sp. strain WT

Abstract

Laccases are multicopper oxidases with various biotechnological applications that oxidize different aromatic or inorganic substrates. In present work, different bacterial strains isolated from Urmia lake, a hypersaline lake in northwest of Iran, were screened to find laccase-producing ones. Spore and an extracellular enzyme from a halotolerant spore-forming bacterium, Bacillus sp. strain WT, showed laccase activity toward typical laccase substrates: syringaldazine and 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate). The extracellular laccase (0.01 U mL⁻¹) decolorized sulphonyl green BLE up to 97% at pH 7.0 after two h incubation at 35 °C, without any addition of mediators. This enzyme with apparent molecular mass of 180 kDa was purified using ammonium sulfate precipitation method and anion exchange chromatography. The optimum laccase activity toward 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine was at 55 °C and pH values of 5.0 and 8.0, respectively. One mM of metal ions, Na⁺ and Ni²⁺, increased the enzyme activity by 12%. The enzyme from Bacillus sp. strain WT could be able to tolerate up to 600–800 mM NaCl (a very strong laccase inhibitor) and showed halotolerant nature with maximum activity at 100 mM NaCl. One mM NaN₃ (another potent laccase inhibitor) almost had no effect on the laccase activity; however, 1 mM l-Cys reduced 87% of its original activity. K_M values for the purified enzyme on 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine were determined to be 132.7 and 3.7 μM, with corresponding k_cat values of 309 and 51 s⁻¹, respectively. The present study is among the first studies on laccase activity of a halotolerant bacterial strain isolated from a hypersaline lake.