Purification and immobilization of laccase from Trichoderma harzianum strain HZN10 and its application in dye decolorization

Abstract

In this study we report the purification of laccase produced by Trichoderma harzianum strain HZN10 (using wheat bran under solid state fermentation) and its application in decolorization of synthetic dyes. Extracellular laccase was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with specific activity of 162.5 U/mg and 25-fold purification. Purified laccase was immobilized in various entrapments like calcium alginate, copper alginate, calcium alginate–chitosan beads and sol–gel matrix. Optimization results revealed that the laccase immobilized in sol–gel was optimally active in wide pH range (4.0–7.0) and thermo-stable (50–70 °C) than free enzyme which was optimum at 50 °C and pH 6.0. Kinetic analysis showed Km of 0.5 mM and 2.0 mM and Vmax of 285 U/mg and 500 U/mg by free laccase and sol–gel immobilized laccase respectively with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] substrate. Free and immobilized laccase was employed for decolorization of three different synthetic dyes (malachite green, methylene blue and congo red). High performance liquid chromatography (HPLC) analysis results revealed that approximately 100% of malachite green, 90% of methylene blue and 60% of congo red dyes at initial concentration of 200 mg/L were decolorized within 16, 18 and 20 h, respectively by laccase immobilized in sol–gel matrix in the presence of 1-hydroxybenzotriazole (HBT) mediator. During the decolorization all three synthetic dyes showed various peaks on HPLC chromatogram indicating different by-products formation. Finally, phytotoxicity analysis results revealed that the by-products of synthetic dyes (formed during decolorization) showed less toxicity against Phaseolus mungo compared to untreated synthetic dyes.

Bioprocess optimization of laccase production through solid substrate fermentation using Perenniporia tephropora‐L168 and its application in bioremediation of triaryl‐methane dye

“…The HPLC elution profiles of native Malachite green showed one peak at retention time 6.68 min whereas laccase treated dye showed the peak at shorter retention time near 4.27 min (Figure ). Such shift in a peak during HPLC analysis of native and treated dye is in accordance with earlier findings . In malachite green degradation a loss of methyl group and formation of leuco‐Malachite green was suggested which resulted in changes in absorption spectra of biotransformed product of dye .…”

“…Dyes are extensively used in textile, paper printing, leather tanning, cosmetics, and pharmaceutical industries . However, these industries could release unutilized dyes as chemical pollutant in their effluents . Most of these dyes are toxic, carcinogenic in nature causing serious environmental concerns .…”

“…15 However, these industries could release unutilized dyes as chemical pollutant in their effluents. 16 Most of these dyes are toxic, carcinogenic in nature causing serious environmental concerns. 17,18 They are also responsible for organ damage, mutation, and development of abnormalities in newborn.…”

Optimization of laccase production and its application in delignification of biomass

“…The fungi was identified as Trichoderma harzianum strain HZN10 on the basis of 18S rDNA gene sequencing and deposited to National Center for Biotechnology Information (NCBI) with GenBank accession number KP050785. SSF process for laccase production by Trichoderma harzianum strain HZN10 was performed in 250-ml glass (Erlenmeyer) flasks consisting 10 g of each material (WB, RS, SB, CC, SD, GS and LL) individually moistened (70%) with the liquid media containing (g/L) (NH 4 ) 2 SO 4 1, CaCl 2 0.125, NaH 2 PO 4-H 2 O 1 and MgSO 4 Á7H 2 O 0.5 without glucose (Bagewadi et al 2017). The inoculum was prepared in the form of mycelial pellets by growing Trichoderma harzianum strain HZN10 in the liquid media on a shaking incubator (150 rpm) at 30 ± 2°C for 4 days.…”

“…The activity of laccase was estimated by the oxidation of 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (0.5 mM) in accordance with the method reported by Bagewadi et al (2017). The changes in absorbance (k max = 420 nm) were recorded at 1-min intervals.…”