I performed pullulase activity from fermented broth of fungi, by using DNS method, but each time I got the reverse result. Can any one suggest me why?
Methods used
0.5 ml filterate+ 0.5ml Pullulan incubated for 15 min at 300C. 1 ml DNS Added, then heated in water bath for 10 min . volume made up to 5 ml by distilled water. taken OD at 570 nm or 540 nm.
Substrate is pullullan and product is glucose
Measuring product conc. using DNS
Result obtained each time
Time O.D.
0 hrs 0.150
24 hrs 0.127
48 hrs 0.106
72 hrs 0.112
96 hrs 0.114
1. Did you add in reaction mixture antibiotic/inhibitor of microorganisms growth? It is possible that appeared sugar was consumed by microbes.
2. Are you sure that your strain produces pullulanase or growth conditions provide enzyme production?
The blank is critical. Generally the blank with enzyme extract could hydrolyze when color of DNS is developed. You can make the DNS reaction (95 or 100 °C) without enzyme extract and when it get cold you can add the enzyme extract. If you do this you can avoid hydrolysis from enzyme extract by temperature.
if you have a suitable protocol for pullulase assay please send it to me.
Thanks
Regards
Megazyme or Sigma sells azo modified substrate for hydrolytic enzymes. Maybe you can change your substrate if they have this substrate. Usually, I perform xylanase activity and sometimes this problem is present, we solve with the blank preparation that I suggest, but also we use this alternative for comparing and validating results.
Try raising the temperature of the assay to 50 °C. Most fungal hydrolytic enzymes act around 50 °C.
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