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  • Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4.

Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4.

PloS one (2014-05-30)
Sonica Sondhi, Prince Sharma, Shilpa Saini, Neena Puri, Naveen Gupta
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 (kcat/Km) 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
p-Phenylenediamine, 98% (GC)
Sigma-Aldrich
Guaiacol, natural, ≥99%, FG
Sigma-Aldrich
Guaiacol, oxidation indicator
Supelco
Guaiacol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
p-Phenylenediamine, sublimed, ≥99%
Sigma-Aldrich
Syringaldazine, indicator for laccase and peroxidase activity
Sigma-Aldrich
p-Phenylenediamine, ≥99.0% (GC/NT)
Sigma-Aldrich
Syringaldazine, 98%
Guaiacol, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Granulocyte Chemotactic Protein-2 human, >97% (SDS-PAGE), recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture