Identification characterization and optimization of bacterial laccase production from Brevundimonas sp mvsp and its potential application in dye degradation

Abstract

The present study deals with the isolation, screening, and characterization of laccase producing bacterial strain from paper and pulp industry waste water. A novel laccase producing bacterial strain Brevundimonas sp. MVSP was identified and the optimum conditions required for maximal enzyme activity was determined. Serial dilution method was used for isolation of microbes from wastewater and identification of laccase producing strain using selective media. Out of fifty-eight isolated microorganisms, seven laccase positive strains were identified. Among the seven isolates, the isolate named MVSP showed highest enzyme activity towards laccase production. The isolated microorganism was characterized by biochemical tests and molecular analysis. The 16s rRNA sequence were analysed and the results was compared with Genbank database. To intensify laccase production, various operating parameters such as incubation time, pH, temperature, carbon and nitrogen sources were optimized. One of the isolated strains produced higher laccase activity, and it was identified as Brevundimonas sp. by 16S rRNA analysis. Its phylogenetic tree was determined and received the Gen-Bank accession number as KP712776. The crude enzyme isolated from Brevundimonas sp. MVSP strain showed the maximum laccase activity of 5.24 U/ml at the optimum conditions. The optimal media composition for novel strain needs to be identified, for enhanced laccase production. The statistical Plackett-Burman designs was used to determine the influence of various parameters in laccase production. The results, showed the significant influence of parameters such as inoculum size, time, temperature, pH and MnSO4 concentration in laccase production. The parameters were further optimized using Box-Behnken design to intensify the laccase production in Brevundimonas sp. MVSP. The statistical optimization process resulted in nine fold increase in laccase activity (45.85 U/ml) by Brevundimonas sp. MVSP. Purification and characterization of laccase enzyme from Brevundimonas sp. MVSP bacteria involve three steps i.e., ammonium sulphate precipitation, Dialysis and DEAE-cellulose chromatography. During the first step using ammonium sulphate precipitation, laccase enzyme yield was 57.2% with 0.9 purification-folds. Then in second step, the enzyme solution was again dissolved with a buffer and sample was kept into dialysis bag for overnight. After the dialysis process, laccase yield 73.4% which showed 1.2 purification-folds. At Final step, the precipitated enzyme was dissolved with a buffer and applied to DEAE-cellulose ion exchange column. The purified enzyme had a peak at DEAE cellulose ion exchange column in the final step, which showed 6.2 purification-folds with a 96.9% yield. After the purification process, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the molecular weight of the bacterial laccase enzyme (isolated from Brevundimonas sp. MVSP) was found to be 43 KDa. The optimal values for enhanced laccase production are as follows: with pH level of 8, temperature (40°C), metal ions (CO2+), inhibitors (NaCl), surfactants (CTAB) and organic solvents (acetone) on enzyme activity were studie newline