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Effect of temperature on enzyme kinetics


Reagent Preparation:


  1. 1% starch solution – 1g of starch in 100ml 0.05M phosphate buffer (pH 6.9). 

  2. 2N NaOH solution -  8g NaOH in 100ml distilled water.

  3. DNS Solution – 1g of DNS was dissolved in 50ml of distilled water. Add 30g of sodium potassium tartarate tetrahydrate in small             lots, the solution turns milky yellow colour. The final volume was made to 100ml with distilled water. This solution was stored in             amber colored bottle.



1. 0.5ml of the substrate solution and 0.5ml of the enzyme solution was pipette into two separate test tubes. Then both the substrate and                the enzyme were preincubated in separate test tubes at 10°C for 5 minutes.
2. After preincubation at that temperature, the substrate solution from the test tube mixed and incubated for 5minutes at that temperature for         the reaction to take place.
3. At the end of the incubation time, 0.5ml of the 2N NaOH was added to arrest the reaction followed by 1.0ml of DNS reagent.
4. The above three steps were repeated to study the effect of temperature at 25°C, 37°C, 55°C and 80°C by preincubation followed by the                incubation at that respective temperature.
5. The control at respective temperature was set up by adding the 0.5ml of the substrate, 0.5ml of 2N NaOH. 
6. After the incubation, all the tubes were placed in a boiling water bath for 5min and cooled to room temperature.
7. The absorbance of all the tubes against respective control was read at 540nm.
8. The amount of product liberated was determined from maltose calibration curve and hence activity at each temperature was obtained.
9. The graph was plotted with activity versus time and optimum temperature was determined.
10. The graph was plotted with the activity versus 1/Temperature in K , the slope of which gives the value of activation E/R.






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