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

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2016-12, Vol.134, p.89-97
• Main Authors: Siroosi, Maryam, Amoozegar, Mohammad Ali, Khajeh, Khosro
• Format: Article
• Language: English
• Subjects: Azo dye; Bacillus; Laccase; Purification; Spore
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2016.10.001

[Display omitted] •Laccase from a halotolerant bacterium showed azo dye decolorization activity.•Up to 400mM NaCl increased purified laccase activity.•Purified laccase showed thermal activation.•Purified laccase was an alkaline laccase.•Purified laccase tolerated NaN3, a common laccase inhibitor. 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 (0.01UmL−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 180kDa 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 600–800mM NaCl (a very strong laccase inhibitor) and showed halotolerant nature with maximum activity at 100mM NaCl. One mM NaN3 (another potent laccase inhibitor) almost had no effect on the laccase activity; however, 1mM l-Cys reduced 87% of its original activity. KM values for the purified enzyme on 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine were determined to be 132.7 and 3.7μM, with corresponding kcat values of 309 and 51s−1, respectively. The present study is among the first studies on laccase activity of a halotolerant bacterial strain isolated from a hypersaline lake.