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enzymes explained

THE ACTION OF AMYLASE ON STARCH

Introduction

The action of an enzyme in speeding the biochemical conversion of a substance into something else can be simply described as follows:
substrate turned into product (enzyme above)

In this investigation, amylase is the enzyme, and starch is the substrate, i.e. the substance it acts upon.
In fact this work should add to your knowledge about the process of digestion which occurs in your body.

This practical work is designed to give you some experience about:

i) how enzymes work, using amylase as an example

ii) what a "control" experiment tells you

iii) how to carry out 2 common food tests


A "control" is a standard experimental treatment which includes all the factors thought to be significant, except one, so that details of its effects may be discovered by comparison.
The "boiled amylase" mentioned below has already been boiled before the class and cooled for your use. Because amylase is a protein (like all enzymes), its molecular structure is affected by this heating process.

Materials provided for class use i.e. shared

AS - amylase solution provided on each bench

BAS - boiled amylase solution in boiling tubes, with plastic pipettes

SS - starch sol

DO NOT CONTAMINATE PIPETTES !

SP - starch powder MP - maltose powder in beakers with own spatulas

GP - glucose powder

DO NOT MIX UP SPATULAS OR REMOVE FROM FRONT BENCH

Reagents and apparatus required - per pair:

iodine solution (I/KI)

Benedict's solution in dropper bottles

8 CLEAN test-tubes

1 test-tube rack

1 glass/plastic rod

1 recessed tile

paper towel

spirit marker

safety glasses

plastic pot

Facilities in lab:

Waterbaths: MAKE SURE YOU KNOW THE DIFFERENCE!

37 C - i.e. "warm" - source of water for incubation

- for transfer to plastic pots on pupils' benches

100 C - i.e. "boiling" - CARE! HOT WATER AND STEAM CAN SCALD!

- for heating - use in Benedict's test

Practical Procedure

(n.b. 1 ml = 1 cm3 - but all apparatus is marked in ml!)

* Points marked with an asterisk are referred to in questions on on these sheets.

1) Take 5 of the tubes, and using a spirit marker*, label them with your own name or mark, plus the numbers 1- 5.

2) Place them in the rack, then, using a graduated plastic pipette, add the various liquids to them as outlined in the tables on the next page. This can be done quite efficiently if you plan your actions beforehand. Make sure you do not contaminate the pipettes.

3) Transfer the 5 tubes to the plastic pot containing water from the "warm" waterbath*, and leave them "for some time". You will be told when to continue with these. Meanwhile carry out the experiment below.

Another experiment to find out about solubility of sugars and starch, and give you practice in the tests for them

4) While you are waiting, use the other 3 tubes for some investigations about starch and the sugars maltose and glucose, and get to know the methods of testing for them:

[Reminder: Benedict's test is for simple (reducing) sugars, and the iodine test is for starch].

a) Label the tubes: "S" - starch, "M" - maltose, "G" - glucose.

Click for a photo b) Collect 1 small spatula measure of the appropriate powders in the tubes, then (using wash-bottle, not pipette) add about 2.5 ml of water to each, and shake. Compare the results.*

c) Place 1 drop of iodine solution into each of 3 recesses in the white tile.

d) Take a clean glass/plastic rod, and insert it into the tube marked "S", then transfer a drop of liquid to one of the iodine drops, and mix round. Note (in the table below) the colour and appearance.*

Click for a photo e) Rinse the rod, wipe it and repeat the process with "M" & "G"

f) Add an equal amount of Benedict's solution to tubes "S" "M" &"G".

g) Place the tubes into the boiling waterbath. CARE !

Click for a photo Check the appearance of these tubes at half-minute intervals. Note the intermediate and record final colour and appearance.*

h) If you like, repeat these tests on the starch and amylase you have been using.*

i) Rinse out these 3 tubes you have been using for food tests.

S - Starch M - Maltose G - Glucose
solubility/result cloudy clear clear
colour with iodine blue-black clear orange clear orange
Benedict's blue orange ppt orange ppt

5) Fetch the 5 tubes from the waterbath, and carry out the same 2 tests on each of them.

Record the results in the table on the next page.

6) Rinse your "wet" apparatus, and put it in the bowl to be washed up. Put away the rest of the apparatus where it came from.

Table for results from main experiment. This is done using your eyes! Click for a photo

Tube number Contents Results after incubation (final colour)
Iodine test Benedict's test
1
2.5 ml amylase soln
+ 2.5 ml starch sol
browny-orange bright orange ppt
2 2.5 ml boiled amylase soln
+ 2.5 ml starch sol
blue-black (hopefully) clear blue
- but could be a little green!
3 2.5 ml amylase soln
orange clear blue ?
4 2.5 ml boiled amylase soln
orange (hopefully) clear blue
- but could also be a little green
5 2.5 ml starch sol
blue-black clear blue

Questions about the "food tests" on the main biochemical substances

How did maltose and starch differ when water was added to them?

> maltose dissolved to give a clear liquid

> starch did not dissolve - stayed cloudy

What (sort of) substance is the iodine test used to identify?

> starch

How specific is this test? i.e. Does it work on one, or lots of other substances?

> very - only works with starch

What is the final colour if the iodine test is:

positive > blue-black negative > browny-orange-red

What (sort of) substance is Benedict's test used to identify?

> reducing sugar

How specific is this test? i.e. Does it work on one, or lots of other substances?

> NOT VERY - same result with glucose, maltose, etc etc.

What is the final colour if Benedict's test is:

positive > orange/murky green negative > clear blue

How did the amylase solution differ (in appearance) from the boiled amylase solution?
> boiled amylase was more cloudy - contained "denatured" enzyme

Click for a photo

Following on from this, complete the next table with your findings - conclusions ( what actual or general substances are present or absent after the enzyme has had a chance to act on its substrate).

Table for conclusions from main experiment. This is done using your brain !

Tube number Contents Amount of substance concluded to be present or absent
(+++, ++, +, -) after incubation - according to
Iodine test Benedict's test
for > starch for> reducing sugars
1
2.5 ml amylase soln
+ 2.5 ml starch sol
- +++
2 2.5 ml boiled amylase soln
+ 2.5 ml starch sol
+++ -/+
3 2.5 ml amylase soln
- -
4 2.5 ml boiled amylase soln
- -/+
5 2.5 ml starch sol
+++ -

What was the purpose of having two separate tables ?

> you could be wrong with your conclusions but the results would still be valid and someone else could interpret them

Questions about the main experiment

In this experiment, you used a plastic pot of water to keep the tubes and their contents "warm".

What do you think would happen if you had not used it? Clue: think about conditions in your body

> slower action if cooler

From your results, what can you say about the product of the action of amylase on starch?

i.e. What sort of substance is it?

> it is a reducing sugar - not shown to be a specific sugar

What was the effect of boiling amylase (on its action with starch)?

i.e. What does boiling do to the enzyme amylase, or

What is the difference between the action of amylase and boiled amylase?

> prevented its normal action
(turning starch into reducing sugar)

Why do you think that starch and maltose were compared in the tests "while you were waiting"?

> these are the substrate and the product respectively of the reaction catalysed by the enzyme amylase

Sometimes, enzymes like amylase contain a small amount of reducing sugar.

If this is so, what will you base your conclusions on?

> differences between tubes 1&3, 2&4

Why were tubes 3 - 5 set up?

> as controls -to see if ingredients in tubes 1 & 2 changed of their own accord

Why did you have to use a "spirit marker" to mark your tubes?

> ordinary ink/marker would wash off in the waterbath

Extra background information

Amylase is a general name for an enzyme which breaks down starch.

Starch makes up a large proportion of our diet, and it needs to be made soluble before it can be absorbed into our bodies. In order to carry out this chemical breakdown, various glands produce digestive juices containing amylases to be mixed with the food. Saliva contains one amylase, and pancreatic juice contains another.
Different amylase enzymes are produced by other organisms, including fungi and bacteria, which carry out external digestion. In fact, any organisms that grow on starch must produce an enzyme to break it down.
Plant seeds often contain a reserve of rather dry insoluble starch for the embryo plant, and when a seed germinates it must produce an amylase to convert the starch into soluble sugars which can be transported to the growing points at tips of root and shoot of the growing plant.
Surprisingly, this is the starting point for making beer: "malting" of germinating barley. Amylases are also important in other phases of brewing: removing starch "hazes" to produce clear "bright" beer.

There are many biotechnological applications involving amylases - see later notes.

What sort of differences would you expect between physical conditions required by the two amylases in our body? What glands produce them?

Amylase produced by:

>salivary glands requires: > neutral conditions

>pancreas requires: > alkaline conditions

Cells at the growing points in a germinating seedling might use sugars for 2 life processes. What are they?

>respiration >growth

What chemical or physical property of starch makes it likely to produce a "haze"?

> insolubility

What other applications of enzymes in everyday life can you find? Try to explain the reasons behind their use. What are the alternatives? Are there any drawbacks?

>biological washing powder - to dissolve products of biological origin on clothes - better than detergent alone (non biological version)? - may cause allergies

> contact lens cleaner

>?




This topic has connections with other units on:-
DIGESTION & ASSOCIATED PROCESSES
THE DIGESTIVE SYSTEM


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