Effect of pH on enzyme activity- amylase

[GaMeR4LyF]

Hi guys, I've got to do a project for school on the investigation of the effect of pH on the enzyme amylase. I am abit lost right now only just started A levels. Can someone suggest how i would carry out this investigation? Equipment and method would be much appreciated! And the results need to be quantitative and not qualitative. so how can i adapt the experiment so i get quantitative results?

 

[chemisttree]

First you need to determine how to measure the activity of the amylase. Amylase converts starch to sugar, so you will need a method to determine the sugar. Find a soluble starch and place it into different pH buffers with the amylase and measure the rate of sugar production.

 

[GaMeR4LyF]

hmm...how about i leave a 1percent starch solution with amylase with buffers of pH 2, 3, 5, 7 and 9 for 5 minutes then use benedicts reagent to test for reducing sugars... those tested positive with reducing sugars would sugest there is enzyme activity... But i don't know how to obtain quantitative results from this...

 

[chemisttree]

You would have to measure the intensity of the color change with the Benedicts reagent (used in excess) to quantify it. Dilution after the color development stage might be required. You can make up a set of standards to compare against and bracket the concentration in that way. Your eye would be the detector. You would measure how much the sample faded from blue to colorless in that example.
Alternatively, you could measure using urine test strips that diabetics use. Should cost roughly a dime per test.

 

[Kushal]

benedict's test can be quite hectic wen you have to heat in boiling water for two minutes each time.

i'd rather test for the remaining starch. you should measure the time for the complete hydrolysis to occur. fo rthat you use a stopwatch, and at say 2 min, intervals(it really depends on the concentration of solution you use), use a dropper and place it on a spot tile, where you previously had placed iodine in the holes.

wen reaction is over, all starch would have been digested, and you will get a brown solution with the iodine. (iodine + starch gives a blue black ppt.)