Prajapati et al.

sample. One unit of enzyme activity (U) was defined as

Co – Cf

the amount of enzyme required to oxidize 1µM ABTS

% D =

_________________

× 100

per min.

Co

2.6.2 Manganese Peroxidase (MnP) assay

Where, Co and Cf are the initial and final concentrations

respectively.

Manganese peroxidase activity (E.C. 1.11.1.13) was

measured by oxidation of DMP (ε = 27,500 M -1 cm -1 ) in

Fungal biomass was determined by weighing the dry

mycelia after decolorization. The mycelial growth was

the presence of H ₂ O and MnSO . The reaction mixture

2

4

squeezed to remove the medium, washed with distilled

contained 1 mM DMP, 0.1 mM H ₂ O , 1 mM MnSO ,

2

4

100 mM Sodium tartarate buffer (pH 4.5) and suitably

water, and dried completely at 60°C till constant dry

diluted sample. Increase in absorbance was measured at

weight. Biomass was calculated by subtracting the weight

469 nm and one unit of enzyme activity (U) was defined

of initial fungal growth from the final weight.

as the amount of enzyme catalyzing the oxidation of 1

HPTLC analysis was performed using precoated silica

µM of DMP per min (Martinez et al . 1996). Presence of

gel 60 F254 plate (Merck, Germany). A 5µl of the

MnP activity was corrected for manganese-independent

control and decolorized samples were spotted on TLC

peroxidase (MnIP) activity by subtracting the activity

plate using micro syringe (HPTLC, Camag, Linomat 5).

obtained at pH 3.25 in the absence of MnSO ₄ at 469

The solvent system used was isopropanol : ethyl methyl

nm.

ketone : liquor ammonia in the ratio of 5:3:2. Developed

plates were dried in air and the dye chromatogram was

2.7 Effect of Physico-chemical factors on decolorization

observed by exposing it to ultraviolet light (254 nm) and

In order to study the effect of various physico-chemical

in visible light using Camag TLC scanner 3.

parameters, the decolorization of Reactive Red M5B was

assayed using different pH (3.0-8.0), temperature (15-

2.9 Data analysis

50 o C) and dye concentration (100-500 mg l -1 ). In the next

All the experiments and the analysis were carried out

step, the effect of various carbon and nitrogen sources

in triplicates and the data presented is the mean value

was analyzed for the effective decolorization of the dye.

of the triplicates. The standard deviation was calculated

using the mean values and remained within the range of

2.8 Analytical measurements

±10%.

Two ml aliquot of decolorized medium was centrifuged

at 10,000 rpm for 10 min and the clear supernatant

3. Results and Discussion

was subjected for the measurement of absorbance at

3.1 Isolation and screening of dye decolorizing fungal

the maximum absorbance wavelength (λ _max ) of the

dyes using UV-visible spectrophotometer (SHIMADZU

cultures

UV-1800, Japan).The residual dye concentration in the

The fungal cultures with an efficient decolorization

sample was determined from the absorbance values

potential of textile azo dyes were isolated from dye

using the calibration curve for absorbance versus dye

contaminated soil samples. It is expected that sites

concentration obtained by plotting the corresponding

near these industries harbor several microorganisms

maximum absorbance in the UV-visible spectra at

which are capable to co-exist with higher toxic levels

different concentrations of dye.

of pollution. Total four fungal cultures were isolated by

The percentage decolorization (% D) was calculated

the enrichment culture technique and were designated as

using the following formula:

AGYP-1, AGYP-2, AGYP-3 and AGYP-4 respectively.

The screening in agar plates is considered as simple and

efficient method to assess the dye decolorization potential

of various microorganisms. All the four isolates were

502