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

The cotA from <i>B. clausii</i>, compared to its ortholog from <i>B. subtilis</i>.

<p>(A) Nearest-neighbor tree of B. clausii cotA as described in the text. The bracketed number is the number of sequences represented by each node. Bootstrap values are given at the base of each branch. (B) The substrate binding pocket of cotA highlighting the differences between <i>B. subtilis</i> (green) and <i>B. clausii</i> (purple) cotA as described in the text. Amino acid changes are numbered on the <i>B. subtilis</i> cotA sequence and annotated with the <i>B. clausii</i> cotA amino acid last. (C) The water exit channel of cotA with similar highlighting. The T2 copper site is visible through the open channel. Small blue spheres represent water from pdb structure 3ZDW. Structures were visualized using PyMol 1.5 and the tree was generated using MEGA 5.</p

Short-term residual activity of the proteins after incubation.

<p>Heat activation is visible at pH(circles, solid line), 9 (squares, broken line) and 10 (triangles, dotted line). (A) <i>B. subtilis</i> cotA at 50°C; (B) <i>B. subtilis</i> cotA at 70°C; (C) <i>B. clausii</i> cotA at 50°C; (D) <i>B. clausii</i> cotA at 70°C.</p

k_cat and K_M for DMP oxidation as a function of pH for <i>B. subtilis</i> cotA (broken lines) and <i>B. clausii</i> cotA (full lines).

<p>(A) k_cat in s⁻¹. (B) K_M in mM (C) k_cat/K_M showing the effect on total pH-dependent activity. The profiles were fitted with a Gaussian using a mean pH of 7.7 and an amplitude of 30.8 for <i>B. subtilis</i> cotA and 8.1 and 4.0 for <i>B. clausii</i> cotA, respectively. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099402#pone.0099402.s001" target="_blank">File S1</a> for full data.</p

Activity vs. pH of the cotA from <i>B. clausii</i> (solid lines) on eight substrates, compared with that of cotA from <i>B. subtilis</i> (broken lines).

<p>Activities are averages with standard deviations from triplicate measurements of initial rates of product formation, corrected for buffer effects (see text) and normalized to 100. (A) ABTS; (B) SGZ; (C) DMP; (D) Caffeic acid; (E) Promazine; (F) Phenylhydrazine; (G) Tannic acid; (H) Bilirubin.</p

Activity of cotA from <i>B. subtilis</i> and <i>B. clausii</i> as a function of NaCl concentration and pH.

<p>(A) <i>B. subtilis</i> cotA; (B) <i>B. clausii</i> cotA; (C) Intersection at 400 mM NaCl and (D) intersection at 1000 mM NaCl, with <i>B. clausii</i> cotA in full lines and <i>B. subtilis</i> cotA in broken lines.</p

Stabilities of the cotAs from <i>B. clausii</i> and <i>B. subtilis</i> vs. temperature and pH 8 (circles, solid line), 9 (squares, broken line) and 10 (triangles, dotted line).

<p>(A) <i>B. subtilis</i> cotA at 50°C; (B) <i>B. subtilis</i> cotA at 70°C; (C) <i>B. subtilis</i> cotA at 80°C; (D) <i>B. clausii</i> cotA at 50°C; (E) <i>B. clausii</i> cotA at 70°C; (F) <i>B. clausii</i> cotA at 80°C. Activities were normalized to the starting activity before heat incubation.</p