Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from <i>Bacillus clausii</i>

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization

A halotolerant laccase from Chaetomium strain isolated from desert soil and its ability for dye decolourization

13 p.-6 fig.-3 tab.A novel fungal laccase produced by the ascomycete Chaetomium sp. isolated from arid soil was purified and characterized and its ability to remove dyes was determined. Extracellular laccase was purified 15-fold from the crude culture to homogeneity with an overall yield of 50% using ultrafiltration and anion-exchange chromatography. The purified enzyme was found to be a monomeric protein with a molecular mass of 68 kDa, estimated by SDS-PAGE, and with an isoelectric point of 5.5. The optimal temperature and pH value for laccase activity toward 2,6-DMP were 60 °C and 3.0, respectively. It was stable at temperatures below 50 °C and at alkaline conditions. Kinetic study showed that this laccase showed higher affinity on ABTS than on 2,6-DMP. Its activity was enhanced by the presence of several metal ions such as Mg2+, Ca2+ and Zn2+, while it was strongly inhibited by Fe2+, Ag+ and Hg2+. The novel laccase also showed high, remarkable sodium chloride tolerance. Its ability to decolorize different dyes, with or without HBT (1-hydroxy-benzotriazole), as redox mediator, suggests that this protein may be useful for different industrial applications and/or bioremediation processes.Peer reviewe