Biology

PART I: INTRODUCTION TO THE ASSAY FOR HYDROLYSIS OF STARCH BY AMYLASE

In this part of the exercise, you will be introduced to the hydrolysis of starch, a reaction catalyzed by amylase and the DNS assay used to measure amylase activity. You set up hydrolysis of 5% starch in vitro, at room temperature (25°C), using fungal amylase . Remember this may not be the optimal temperature for this enzyme. ACTIVITY OF AMYLASE IS DIRECTLY RELATED TO THE AMOUNT OF MALTOSE PRODUCED.

EXPERIMENTAL SETUP:

1. Two tubes are prepared according to the table below. For a video on how to use micropipettes, click here:

Tube	Water (μl)	5% Starch (μl)	Fungal Amylase (μl)	Total Volume (μl)
A	450	450	100
B	550	450	0

2. Both tubes are incubated for 10 min at 25°C (on bench).

3. 1000 μl (1 ml) DNS is added and the tubes are placed in boiling water for 5 min.

4. 8 ml of dH2O (deionized water) is added to both samples to dilute them, using a serological pipet. The absorbance of light at 540 nm is read for the two solutions using a spectrophotometer. (Do we need this? It will be in Lab 1)Here is the link to a video on how to use a spectrophotometer like the one we have in our lab:

https://youtu.be/u3C6QXD8gjQ

Tube B is used as a “blank”, to calibrate the machine.

What treatment is tube B in the experiment? _________________________

What treatment is tube A in the experiment? _________________________

5. The absorbance of tube A you read in the spectrophotometer is 0.358 .

6. After completing Part II, the maltose standard curve, determine the maltose concentration in your tube ________________.

PART II: STANDARD CURVE FOR ESTIMATION OF MALTOSE

The objective of this part of the lab activity is to collect data and plot a standard curve. The procedure for this activity is described below:

DNS is added to known amounts of maltose, and the absorbance of the reduced DNS is measured at 540nm, using a spectrophotometer. A reference graph, called a standard curve, is made from this data. A standard curve is a graph which shows a relationship between two quantities; in this activity, the relationship between known concentrations of maltose and their absorbance. You may want to review the “line of best fit” section in the graphing skills part of Exercise 1. In part III of this lab exercise, you will use the standard curve to estimate the amount of maltose produced during hydrolysis of starch, the reaction catalyzed by amylase. The data on the amount of maltose produced will then be used to calculate amylase activity.

To prepare the standard curve, the concentration of maltose (mg/ml) in each tube has to be determined first. Please watch this video https://youtu.be/KNMz0pSgYbk to understand the steps involved in calculations, and to construct the standard curve.

1. Complete the table below and show a sample of your calculations. You will be using the formula CiVi = CfVf (equivalent to C1V1 = C2V2).

The initial maltose concentration (Ci) used is 2.5 mg/ml. After adding 1 ml DNS to 1 ml of maltose, all samples are diluted with 8 ml of water (do you want to mention about the redox reaction occurring at a higher temperature?), and their absorbance is determined in a spectrophotometer and indicated in the table. The final volume of maltose (Vf) is, therefore, 10 ml.

Table 1: Final Concentration of maltose

Volume of initial maltose solution Vi (μl)	Volume of initial maltose solution Vi (ml )	Volume of water added to get a total of 1 ml	Final Maltose Concentration (mg/ml)	Absorbance at 540nm
0				0.000
200				0.228
400				0.487
600				0.751
800				1.031
1000				1.275

2. Graph 1: graph the Absorbance vs. Concentration of maltose from Table 1. There are two ways to do it, a) manually, on graph paper, OR b) in Microsoft Office Excel, depending on what your instructor has asked you to do. Remember to use TAILS and insert your graph below. If you are not sure how to do that, watch the video on maltose standard curve for help. https://youtu.be/KNMz0pSgYbk

Choose one of these two ways to present your graph:

a. Construct your graph manually, on graph paper. Draw a line of best fit through your points, making sure that line passes closer to as many data points as possible. Embed a picture below. This graph will be used in Part III, to determine the amount of maltose produced in your experimental tubes.

OR

b. Construct your graph on excel, according to the instructions on the video provided on the maltose standard curve. Draw a ‘line of best fit’ through your data points by clicking on a point and selecting “add trendline”. In the menu that opens, select “display equation of line, to get the equation of the line to appear on your graph. Embed your excel graph below. You will use this equation of the line in Part III, to determine maltose produced in your experimental tubes.