Cellulase activity was evaluated using carboxymethyl cellulose
(CMC) as substrate for enzymes present in the culture
supernatant [25]. Reagent used were: 50 mol m3 acetate
buffer at pH 5.0; 2% CMC (sigma cat. no. C5678) solution prepared
in the acetate buffer; 0e30 mol m3 glucose standards
prepared in acetate buffer and DNSA reagent (10 kg m3 3,5-
dinitrosalicylic acid, 16 kg m3 sodium hydroxide, 300 kg m3
potassium sodium tartrate tetrahydrate). Samples (1 cm3)
were centrifuged in 1.5 cm3 tubes at 15 000 g for 10 min,
0.5 cm3 of the supernatant was transferred in 4.5 cm3 of 2%
CMC solution, placed in 60 C water bath. Every 10 min,
starting at 0 min after mixing the supernatant and CMC solution,
1 cm3 of the mix was collected and mixed with 3 cm3
DNSA reagent, boiled 5 min, placed in a cold water bath for
3 min. The absorbance at 540 nm was then read on UVevisible
Varian Cary 50 bio spectophotometre. As a blank, 0.1 cm3 acetate
buffer was mixed with 0.9 cm3 CMC solution and 3 cm3
DNSA reagent. The standard curve was made with 0.1 cm3 of
0e30 mol m3 glucose standards, mixed with 0.9 cm3 CMC
solution and 3 cm3 DNSA reagent. The slope of the resulting
curve gave the CMC-ase activity. The cellulolytic activity of the
supernatant will be shown in enzyme unit (U), which is the
amount in mmole of reducing sugars released per minute