Purification and Characterization of an Extracellular, Thermo-Alkali-Stable, Metal Tolerant Laccase from Bacillus tequilensis SN4

Abstract

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k cat/K m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu2+, Co2+, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.

Overexpression of a Laccase with Dye Decolorization Activity from <b><i>Bacillus</i></b> sp. Induced in <b><i>Escherichia coli</i></b>

“…5 a), which is similar to the findings of many researchers who demonstrated that the optimal temperatures of laccases are between 30 and 80 ° C Sondhi et al, 2014;Telke et al, 2011]. Furthermore, the recombinant laccase showed a high thermostability with a half-life of about 15.9 h at 70 ° C. The half-life of inactivation at 70 ° C was much higher than some other Bacillus strains, such as 6.9 h at 70 ° C in LS04 [Lu et al, 2013] and 3.5 h at 65 ° C in DSM 27 [Reiss et al, 2011], and was similar to those from other Bacillus strains [Guan et al, 2014;Sondhi et al, 2014]. The higher stability of the enzyme might be adapted to cope with the industrial process requirements due to longer stability under high temperatures.…”

“…The purified laccase showed the lowest K m values (31.2 μ M ) for oxidizing ABTS when compared to the other tested substrates, and the K m value was also significantly lower than other Bacillus strains, which fell in the range of 44-1,404 μ M [Lu et al, 2013;Mohammadian et al, 2010;Sondhi et al, 2014], indicating that the laccase from Bacillus sp. A4 has a much higher affinity for ABTS substrate.…”

Characterization of a Novel Laccase-producing Bacillus sp. A4 and its Application in Miscanthus Degradation

“…Lorenzo et al (2005) reported that the addition of Cu 2+ to the reaction mixture stimulated laccase activity at concentrations lower than 1.0 mM and that Mn 2+ and Zn 2+ did not affect enzyme activity at any of the concentrations tested (0.5 mM to 80 mM). The filling of the Type I Copper binding site by Cu 2+ could be the main cause of this increased laccase activity (Shekher et al 2011;Sondhi et al 2014). Purified laccase activity was induced 5% to 10% methanol and 10% to 20% ethanol (Fig.…”

“…4), which was different from most of the reported laccases of bacteria and fungi, though several isoforms of laccases from bacteria have been reported to range from 31 kDa to 40 kDa in size. For example, Sondhi et al (2014) found a novel extracellular thermo-alkali-stable laccase of Bacillus tequilensis SN4 that produced a 32 kDa monomeric protein. The molecular weight of laccase from Streptomyces coelicolor was around 32 kDa and able to rapidly decolourize dye in the presence of syringaldehyde, which acted as a redox mediator (Dubé et al 2008).…”

Heterologous expression of an acidophilic multicopper oxidase in Escherichia coli and its applications in biorecovery of gold

“…Recently, Sondhi et al reported that the strain Bacillus tequilensis SN4 had the highest catalytic activity of MCO (i.e., k cat is 4020/min) at pH 5.5 and 85 °C (Sondhi et al 2014). As shown in Table 2, the reaction conditions of MCO activity are very diverse among different microbes (Ye et al 2010;Ausec et al 2015;Yang et al 2016;Safary et al 2016).…”