The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Abstract

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209-216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.

Keywords: Bacillus subtilis; Guaiacol; Isolation; Laccase; Pulp and paper wastewater.

Fig. 1

Screening of isolated strains on Petri plates containing nutrient agar medium. aBacillus subtilis WPI on nutrient agar medium without guaiacol; bB. subtilis WPI on nutrient agar medium containing guaiacol

Fig. 2

PCR products of 16S rDNA from Bacillus subtilis WPI (1500 bp). Lane L: molecular weight marker 1 KB (Fermentas, UK); Lane S: B. subtilis WPI

Fig. 3

Phylogenetic tree of 16S rDNA sequence of Bacillus subtilis WPI strain and related taxa

Fig. 4

The assay solution with different substrates. a the assay solution with ABTS (a) before adding ABTS, (b) after adding ABTS. b the assay solution with guaiacol (a) before adding guaiacol, (b) after adding guaiacol

Fig. 5

SDS-PAGE and Native PAGE of the cell extract of Bacillus subtilis WP1. Lane 1 Pre-stained size marker of protein Lane 2B. subtilis WPI cell extract SDS-PAGE. Lane 3: laccase activity was detected by activity staining with ABTS in succinic buffer (pH 5.0). The loaded samples on the gel were the soluble proteins released after sonication (for more details please see the “Materials and methods” section)