Molecular study of Pigeonpea

enzymes production by isolate AGYP-1 was evaluated.

dyes (Reactive Red M5B, Acid Green GLW and Reactive

Decolorization and degradation of Reactive Red M5B

Violet 5R) were inoculated with 10 ml of soil suspension

was studied by UV-Visible spectrophotometric and

(10 % w/v) and incubated under shaking condition (120

HPTLC analysis.

rpm) at 30 o C for 2 weeks. Repeated transfers in fresh

dye containing media were performed till stable dye

2. Materials and methods

decolorizing fungal cultures were obtained. From the

decolorized flasks, fungal mycelia were transferred to

2.1 Dyes and chemicals

MEA for further purification.

Commercially available synthetic textile dyes, Reactive

Red M5B (C.I. Reactive Red 2), Acid Maroon V (C.I.

2.4 Dye decolorization and enzyme screening on solid

Acid Red 119), Reactive Red BS (C.I. Reactive Red 111),

media

Acid Red F2R (C.I. Acid Red 151), Acid Red 3BN (C.I.

Plate assay was performed to detect the decolorization

Acid Red 131), Reactive Blue 3R (C.I. Reactive Blue

activity of fungal isolates. The MM agar plates containing

28), Reactive Violet 5R (C.I. Reactive Violet 5), Acid

individual dye (100 mg l -1 ) were inoculated with a

Red BB (C.I. Acid Red 128), Acid Green GLW (C.I.

mycelial disc (8 mm diameter) from previously grown

Acid Green 27) and Reactive Red HE8B (C.I. Reactive

fungal cultures and incubated at 30 o C for 10 days. The

Red 152) used for present study were procured from

plates were observed for the clearance of dye surrounding

CAB Chemicals, Ankleshwar, Gujarat, India. ABTS (2,

fungal growth. The production of ligninolytic enzymes

2-Azino-bis

(3-ethylbenzthiozoline-6-sulphonic

acid)

was checked on SDA plate containing ortho-dianisidine

was purchased from Sigma (Sigma St. Louis, MO, USA),

(0.01% w/v). Uninoculated MM agar and SDA plates

2, 6-Dimethoxyphenol (DMP) and ortho-dianisidine

were served as control.

were purchased from Lancaster (Lancs, UK) and CDH

(Mumbai, India) respectively, Malt extract agar (MEA)

2.5 Decolorization studies in liquid medium

was procured from Hi-Media Labs (Mumbai, India). All

The dye decolorization experiments were carried out in 250

other chemicals used were of the highest purity and of

ml Erlenmeyer flasks containing 100 ml MM. The flasks

analytical grade.

were inoculated with 8 circular discs (8 mm diameter) cut

2.2 Nutrient media

from actively growing fungal cultures on MEA agar plate

and incubated under shaking condition (120 rpm) at 30 o C.

Mineral Medium (MM) containing g l -1 : glucose,

Upon fifth day of incubation, an individual dye (100 mg

5.0, KH ₂ PO ₄ , 1.0, MgSO 4 .7H 2 O, 0.5, KCl, 0.5, yeast

l -1 ) was added and the flasks were further incubated at

extract, 0.5, and pH 5.0 ± 0.5 was used for isolation and

the same conditions for decolorization. In another set,

decolorization experiments. The fungal cultures were

the flasks were kept in static condition to check its effect

routinely transferred and maintained on malt extract agar

on decolorization. Aliquots were withdrawn periodically

(MEA) containing g l -1 : malt extract, 20.0, agar-agar,

from different flasks and were analyzed for decolorization

30.0, and pH 5.0 ± 0.5. The screening of ligninolytic

and enzyme production.

enzymes was performed on Sabaroud’s dextrose agar

(SDA) containing g l -1 : peptone 10.0, glucose 40.0, agar-

2.6 Determination of enzyme activities

agar 30.0, and pH 5.6 ± 0.5.

2.6.1 Laccase assay

2.3 Isolation of dye decolorizing fungal cultures

Laccase (E.C. 1.10.3.2) activity was determined by

The potential dye decolorizing fungal strains were isolated

monitoring change in absorbance at 420 nm related to

from contaminated soil samples obtained from different

the rate of oxidation of 1 mM ABTS (ε = 36,000 M -1

sites of common effluent treatment plant (CETP) -

cm -1 ) following the method described by Niku-Paavola et

Nandesari - Vadodara, Gujarat, India. 250 ml Erlenmeyer

al . (1990). The assay system contained 1 mM ABTS, 100

flasks containing 100 ml MM and 100 mg l -1 mixture of 3

mM sodium acetate buffer (pH 5.0) and suitably diluted

501