Team:VIT Vellore/Characterization

Team VIT Vellore/Description

Part number: BBa_K523006

E.coli periplasmic α amylase gene malS under control of lac promoter

An Overall Workflow

Obtaining the cell lysate

After preparing competent cells and transforming them, we let the culture incubate at 37°C overnight. The next day, we lysed the cells using ultrasonication, spun it down to remove the debris, and used 0.5ml of the lysate for our experiments. DNS assay enzyme incubation conditions were changed as required.

Note: All enzyme activity are in µg/(ml2*min)

Standard curve for DNS assay

Dinitro salicylate is an oxidizing agent which converts glucose to gluconic acid. Glucose is formed by digesting starch by amylase. We can quantify the activity and calculate the concentration of the amylase produced by correlating the readings with the standard graph.

Standard curve, DNS assay
Conc. of glucose (µg/ml) Absorbance @ 540nm
0 0
200 0.2045
400 0.4845
600 0.7860
800 0.8582
1000 1.198

y = 0.0012x - 0.0009

Media optimization: pH

Checking the most optimum pH for the expression of the part is very important. We performed DNS assay for the cell lysate incubated during reaction time at the pH 3, 5, 6 ,7, 8 using phosphate buffer adjusted at different pH.

Media optimization: pH
pH O.D. Activity
3 0.4352 36.342
4 0.5796 48.375
5 0.6756 56.375
6 0.6532 54.508
7 0.7717 64.383
8 0.5344 44.608
9 0.2971 24.333

As observed, 7 is the optimum pH for amylase production.

Media optimization: Nitrogen source

We used different nitrogen sources to find out which was the most optimum for amylase synthesis. We used yeast extract, beef extract, malt extract and peptone for our experiments.

Media optimization: Nitrogen source Nitrogen Source Activity
1 Yeast extract 1.633
2 Malt extract 0.908
3 Beef extract 14.492
4 Peptone 1.183

As we can clearly see, beef extract is the most optimum nitrogen source for amylase expression.

Media optimization: Carbon source

Along with the nitrogen source, carbon source used heavily impacts the amylase production, as we saw form our experiments.

Media optimization: Carbon source Carbon Source Activity
1 Dextrose 0.433
2 Starch 3.591
3 Sucrose 7.483
4 Fructose 4.867

We can thus conclude that Sucrose is the optimum carbon source for amylase expression.

Characterization of the activity of α amylase:

We decided to test the activity of the enzyme produced by this part. To test this, E.coli cells were transformed with the mentioned part.

Effect of pH

To determine the effect of pH on the activity of the enzyme, the cells containing the part was lysed using ultrasonication, the cell debris was spun down and the supernatant was collected. The supernatant contains the enzyme. The pH of this solution was adjusted to the desired value using the following buffers:

Desired pH Buffer used
3 Glycine – HCl buffer
4 Acetate buffer
5 Acetate buffer
6 Mcllavine buffer (Citrate-Phosphate)
7 Phosphate buffer
8 Phosphate buffer
9 Glycine – NaOH buffer

DNS assay was performed to these solution using the protocol mentioned above.
The following results were obtained:

Effect of pH
pH Activity (µgml-2 min-1)
3 57.48
4 59.53
5 62.33
6 70.66
7 91.51
8 41.67
9 52.54

It is clearly evident pH 7 is the ideal condition for the enzyme to function. At pH even slightly higher than this, a sharp drop in activity is observed. Even at a lower pH, activity remains significantly low.

Effect of temperature

Similar to the previous experiment, cell lysate was collected and incubated in temperatures ranging from 0° to 80° Celsius. Then DNS assay was performed to these samples using the same protocol.


Effect of temperature
Temperature (°C) Activity (µgml-2 min-1)
0 52.12
20 90.8
40 84.91
60 74.47
80 65.2

Maximum activity was observed at 24°C with the activity progressively degrading as the temperature increases. The dip in activity is steeper as the temperature drops but not as steep as we initially expected. The pH used here was 7 and the activity at 24°C is close to the value we obtained from the previous experiment.

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