I have an isolate that shows amylase activity on a starch azure plate incubated at 70°C but when I grew this isolate in broth, centrifuged it, and took its supernatant for performing assays, there was no activity detected by the DNSA method however the same supernatant showed activity on the starch azure plate. I checked its activity at 60°C, 70°C, 80 °C, and 90°C but got no positive results. I tried to change one parameter at one time, i.e. temperature, pH, time, but still I am not getting any activity.
Concentrate (lyophilization) your supernatant into small volume then do the assay.
I also performed assays after concentrating the supernatant still no activity......and when i am adding same concentrated sup on substrate plates it is showing activity ....
I understand that your isolate showed amylase activty when it grows on a starch azure-plate, incubated at 70°C. The culture supernatant also showed amylase activity on starch azure plate, but no activity with DNSA method. Did you use the same incubation time for both methods? if activity on starch azure, takes long time to be detected, and if your assay is not carried (sterile conditions) with a free cell supernatant, take care no get amylase producer microorganisms as contaminant. Looks like yours DNSA method is nort working, Check your DNSA method, use a positive control in order to verify starch hydrolysis. Increase incubation time, use soluble starch, increase volume of supernatant in reaction: in a tesd tube (with cap)add 500 uL of 1% Soluble starch (at working pH, could pH of cultivation) + 500 ul of culture supernatant, 1 h incubation at tested temperature, finish reaction with 200 uL of 2N sodium carbonate, add 1.5 mL of DNS reagent, water bath at 85-100°C for 15 min. a dark solution can be dituted if necesary to read at 550 nm at the sppectrofotometer. You can use a glucose standard in order to check that your reagents are detecting free reducing sugars. Also in your cultivation you could increase amylase production including starch as a carbon source (1%) in the culture media. The normal media, can be decreased in carbon source 1:10 and adding starch as carbon source. Hector Nolasco, CIBNOR
Thank you for answering sir......
My DNSA method is working as i checked it with standard (thermostable amylase as positive control) and found starch hydrolysis . Incubation time for both is different as culture takes some time to grow so activity can be seen after its growth but when i am taking sup.it is showing activity after 3-4 hours and i am performing assay with same sup but with less incubation time ( i checked upto 2 hrs incubation at 70 C and there was no activity ). Also i want to tell u that i am using soluble starch(1%) but i am not performing assays in test tubes, i am doing it in small volumes in ul (as i did with positive control ). I am using maltose standard.
Seems you have a low amylase activity. Concentrate supernatant with a YM10 membrane by ultrafiltration, then use the concentrate for amylase activity. Can you explain how is DNSA method you used?
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