Kingdom of Saudi Arabia

King Abdulaziz University

Girls Collage of Science

Biochemistry Department

Enzymology
Practical Manual
BIOC231

Name:
Computer No.:
Section:

1
 Contents

Lab # Experiment Page

1 Effect of Amylase activity on Starch 3

2 Determination of α-amylase activity 8

3 Effect of pH on amylase activity 12

4 Investigation effect of temperature on the activity of lipase 15

5 Hydrolysis of sucrose by yeast β-Fructofuranosidase 19

Determination of Hydrolyzed Sucrose Solution by

6 benedict quantitative method 23

7 Estimation of lipase activity 27

8 Indirect estimation of lactate dehydrogenase 30

9 Detection of Enzymes 33

10 Detection of Enzyme mixture 36

2
 Experiment 1:Effect of Amylase activity on Starch

Definition of enzymes: enzymes are biological catalysts. They greatly

enhance the rate of specific chemical reactions that would occur
very slowly.
Starch which is the storage form of glucose in plant. Starch consist of

1- Amylose

1-4 α- glycosidic linkage

2- Amylopectin

1-6 α- glycosidic linkage

3
Contents of Saliva:

In animals, saliva is produced in and secreted from the salivary

glands. It is a fluid containing:
 Electrolytes: (2-21 mmol/L sodium, 10-36 mmol/L
potassium, 1.2-2.8 mmol/L calcium, 0.08-0.5 mmol/L
magnesium, 5-40 mmol/L cloride, 2-13 mmol/L bicarbonate,
1.4-39 mmol/L phosphate)
 Mucus. Mucus in saliva mainly consists of
mucopolysaccharides and glycoproteins;
 Antibacterial compounds (thiocyanate, hydrogen peroxide,
and secretory immunoglobulin A)
 various enzymes. The major enzymes found in human saliva
are alpha-amylase, lysozyme, and lingual lipase. Amylase
starts the digestion of starch before the food is even
swallowed. It has pH optima of 6.7-7.4. Human saliva
contains also salivary acid phosphatases A+B, N-
acetylmuramyl-L-alanine amidase, NAD(P)H
dehydrogenase-quinone, salivary lactoperoxidase,
superoxide dismutase, glutathione transferase, glucose-6-
phosphate isomerase, and tissue protein. The presence of
these things causes saliva to sometimes have a foul odor.
Healthy people produce about 1.5 L of saliva per day.

Amylase:

found in two forms:

1. α-amylase (in saliva and pancreatic juice) which is

endoglycosidase that attack starch randomly. Inactivated by the
acidity of the stomach.
2. β-amylase (from plant origin) which is exoglycosidase cleaves
maltose from the non-reducing end to produce β-maltose

4
Principle:

When we want to measure enzyme activity either we measure the

decrease in the substrate concentration or the increase in the product
concentration.

E
[S] [ES] [P]
Amylase
Starch Maltose

pH 6.4-7.2 Cl+ reducing sugar

Indicator I2 Indicator Fehling

Blue color Red copper oxide ppt

Other uses of amylase in industry:

It is used in clarification of fruit juices. The turbidity present in natural
beverages is due primly to the presence of starch and cellulose
molecules too large to be completely soluble. Amylase hydrolysis these
molecules to glucose which are more water soluble.

Reagents:
 Starch 1% solution in 0.3% aqueous sodium chloride
 Freshly prepared; iodinated potassium iodide solution.
 Amylase

5
Procedure:

Prepare 2 test tubes which contain the following:

Test tube A B
Amylase - 1 ml
Starch 1 ml 1 ml
Allow the tubes to stand for 30 min in water bath (37°C - 40°C)
Iodine solution 1-2 drops 1-2 drops

References:

1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 1978

2- Harvey,R and Champe,P. Lippincott biochemistry, london.2005.

6
Results Sheet

Experiment Observation Comment

A
( Starch only)

B
(Starch +
Amylase)

7
 Experiment 2: Determination of α-amylase activity
History:

α-Amylases (EC 3.2.1.1) is an enzyme of glycoside hydrolases mainly

produced in the salivary glands and pancreas, play a well-known role in
hydrolyzing a-1,4- glucosidic bonds between glucose in starch ( consists
of two types of polysaccharide amylose, amylopectin) and maltose is
release. Elevated level of α-Amylases in serum can be used as markers
for clinical diagnosis of diseases, e.g. Pancreatitis. When the pancreas is
diseased or inflamed, amylase releases into the blood.

Principle:

The α -amylase activity is measured using a colorimetric method with

3,5-dinitrosalicylic acid (DNS) reagent. In this method, starch by α –
amylase is converted into maltose. Maltose released from starch is
measured by the reduction of 3,5-dinitrosalicylic acid.

Starch + H2O α-Amylase Maltose (reducing agent)

8
Maltose reduces the pale yellow coloured alkaline 3, 5-Dinitro salicylic
acid (DNS) to the orange- red colored. The intensity of the color is
proportional to the concentration of maltose present in the sample.

This intensity change in color is measured using a spectrophotometer as

the absorbance at 540nm wavelength. Wave length is set to 540 nm
because it is the region where orange-red color absorbs.

This procedure applies to all products that have a specification for α-

amylase

Reagents:
 0.02 M Sodium phosphate buffer
 1% Starch
 2 N Sodium hydroxide
 Sodium potassium tartrate tetrahydrate
9
  Dinitrosalicylic acid color reagent
 Standard Maltose Stock Solution
 Amylase enzyme

Procedure:

Adjust spectrophotometer at 540 nm and 25°C.

Tube Test Blank Standard

Starch 0.5 ml 0.5 ml -
Enzyme 0.5 ml - -
Distell water - 0.5 ml 0.5 ml
Maltose - - 0.5 ml
Mix well and incubate at 25°C for 3 minutes
Dinitrosalicylic
acid color 1 ml 1 ml 1 ml
reagent
Incubate all tubes in a boiling water bath for 5 minutes. Cool to room
temperature
Distell water 10ml 10ml 10ml
Mix well and read the increase in optical density at 540 nm against
blank

Determine micromoles maltose released from standard or standard curve

Calculation

Enzyme activity = OD (test) x concentration of St (µmoles) x dilution of enzyme

OD (st) x incubation time (3 min)

Enzyme Unit = µmoles maltose formed / min/0.5 ml (x 2)

= µmoles maltose formed / min/ml

10
Results Sheet

11
 Experiment 3:Effect of pH on amylase activity
- The effect of pH on α-amylase activity will be studied
- Enzymes are affected by changes in pH. The optimum pH value
is defined as the pH at which the enzyme rate of reaction (enzyme
activity) reach the maximum activity (V max)
- Deviation in pH from the optimum cause decrease in enzyme
catalytic activity
- Extremely high or low pH values generally result in complete loss
of activity for most enzymes.
- The enzyme stability is depending on the optimum pH . Each
enzyme has a region of optimum pH for stability.
- The optimum pH can be determined by incubating the enzyme in
different incubation media containing different pH buffer
range from 1.5 –10. The enzyme activity will be calculated at each
pH at which the enzyme will be incubated. Plot a curve of enzyme
rate of reaction (enzyme activity) against the different pH at
which the enzyme catalytic reactions are incubated.
- From the curve, the optimum pH which give the maximum
activity of the enzyme will be determined

12
 Procedure:

Tube 1 2 3 Blank Standard

Starch (PH=1.5) 0.5 ml - - 0.5 ml -
Starch (PH=6.9) - 0.5 ml - - -
Starch (pH=10) - - 0.5 ml - -
Enzyme 0.5 ml 0.5 ml 0.5 ml - -
Distill water - - - 0.5 ml 0.5 ml
Maltose - - - - 0.5 ml
Mix well and incubate at 25°C for 3 minutes
Dinitrosalicylic
1 ml 1 ml 1 ml 1 ml 1 ml
acid color reagent
Incubate all tubes in a boiling water bath for 5 minutes. Cool to room
temperature
Distill water 8 ml 8 ml 8 ml 8 ml 8 ml
Mix well and read the increase in optical density at 540 nm against
blank

Determine micromoles maltose released from standard or standard curve

Calculation

Enzyme activity = OD (test ) x concentration of St (µmoles) x dilution of enzyme

OD (st) x incubation time (3 min)

Enzyme Unit = µmoles maltose formed / min/ 0.5 ml enzyme (x 2)

= µmoles maltose formed / min/ ml enzyme

13
Results Sheet

14
 Experiment 4:Investigation effect of temperature on the
activity of lipase

This practical gives you a chance to:

 investigate how lipase activity changes with temperature
 consider how indicators can help us to follow chemical reactions.
Procedure
1- Label a test tube with the temperature (25°C- 40°C-
70°C).
2- Add 5 drops of phenolphthalein to the test tube.
3- Measure out 5 ml of milk using a measuring cylinder (or
syringe) and add this to the test tube.
4- Measure out 7 ml of sodium carbonate solution using
another measuring cylinder (or syringe) and add this to
the test tube. The solution should now be pink.
5- Place a thermometer in the test tube. Take care as the
equipment could topple over.
6- Place the test tube in a water bath and leave until the
contents reach the same temperature as the water bath.
7- Remove the thermometer from test tube and replace it
with a glass rod.
8- Use the 2 ml syringe to measure out 1 ml of lipase from
the beaker in the water bath for the temperature you are
investigating.
9- Add the lipase to the test tube and start the stop clock/
stopwatch.
10- Stir the contents of the test tube until the solution loses
its pink color.
11- Stop the clock/ watch and note the time in a suitable
table of results.

Reference:
http://www.nuffieldfoundation.org

15
Results Sheet

Experiment Observation Comment

A (at 25°C)

B (at 40°C)

C (at 70°C)

16
 QUESTIONS

1- When fat breaks down, what is produced?

2- Use this information to explain why the phenolphthalein changes
colour?
3- What is the effect of temperature on the time taken for lipase to
break down the fat in milk?
4- Why does the temperature affect the action of lipase in this way?
5- What is the difference between a ‘time taken’ and a ‘rate of
reaction’ curve for this investigation?

17
ANSWERS

18
 Experiment 5: The hydrolysis of sucrose by yeast β-
Fructofuranosidase

Principle:
β-Fructofuranosidase is a glycosidase found in yeast. It catalyses the
hydrolysis of sucrose to glucose and fructose. The enzyme is also known
as invertase or sucrase, but these names are no longer used.
The substrate sucrose is a non-reducing sugar, whereas the products
formed are both reducing sugar. Therefore the reaction can be followed
by the estimation of the quantity of reducing sugar formed. Between the
several methods which can be used for such estimation, Benedict
quantitative method was utilized.

Benedict quantitative reagent is composed of:

1- Copper sulphate: to provide the oxidizing Cu+2ions.
2- Sodium carbonate: to provide the alkaline medium necessary for
the formation of the highly reactive reducing sugar 1-2 endediol.
3- Sodium citrate: combines with Cu carbonate to prevent its
precipitation by forming a slightly soluble complex with cupric
ions (Cu+2ions). This complex dissociates slowly to give a
sufficient supply of Cu+2 ions.
4- Potassium thiocyanate (KSCN): reacts with cupric ions to give
Cu(SCN)2
5- Potassium ferrocyanide (K4Fe(CN)6): prevents the re-oxidation of
the formed cuprous thiocyanate (CuSCN) to cupric thiocyanate.

19
The reaction takes place as fallow:
1- The enolization of reducing sugar in alkaline medium to give a
highly reactive reducing compound, which is 1-2 endediol

2- Formation of cupric carbonate

Na2CO3 + CuSO4 Cu CO3 + Na2SO4

3- Formation of sodium cupric citrate complex:

20
4- Ionization of sodium cupric complex

5- Reaction of KSCN with Cu+2ions:

Cu+2 + KSCN Cu(SCN)2

Cupric thiocynate (blue)

6- Reduction of Cu(SCN)2 by 1,2 enediol to cuprous thiocynate:

boil
Cu(SCN)2 CuSCN

21
Procedure:
A- Hydrolysis of sucrose by yeast β-Fructofuranosidase
Prepare five tubes containing the following mixtures:

Tube 1 2 3 4 5

Sucrose 0.3M 10 ml 8 ml 6 ml 4 ml 2 ml

D.W 0 2 ml 4 ml 6 ml 8 ml

Buffer pH 4.5 6 ml 6 ml 6 ml 6 ml 6 ml

Pre-incubate at 37C˚ for 5 min

Yeast
4 ml 4 ml 4 ml 4 ml 4 ml
suspension
Incubate for 15 min
1% NaOH 2 ml 2 ml 2 ml 2 ml 2 ml
Final conc. Of
150 120 90 60 30
sucrose

Note:
The yeast must be added to each tube at a constant time intervals, i.e. tube
1 at time 0, tube 2 at 2min etc. This will enable the incubation time to be
measured exactly and ensures that each tube is incubated for the same
time. Incubate each tube for exactly 15 min. Stop the reaction by the
addition of 2ml of 1% sodium hydroxide. This will be at 15 min for tube
1, 17 min for tube 2 and so on.

22
Experiment 6: Determination of the hydrolyzed sucrose solution by
Benedict method

1- Place the sugar solution of hydrolyzed sucrose from the previous

experiment in burette.
2- Measure 5ml of Benedict quantitative reagent into 100 ml conical
flask add approximately 1g of anhydrous sodium carbonate and
few pieces of porcelain. Heat the mixture vigorously.
3- Run in the sugar solution slowly from the burette until a bulky
white precipitate is formed. Continue the titration by adding the
sugar solution drop by drop until the last trace of blue or green has
disappeared.
4- Record the volume of the sugar required to titrate 5 ml of benedict
reagent. This volume will be your titer number.

Note:
1- The end point must be determined while the mixture is still boiling.
When the mixture is not boiling atmospheric oxidation occurs and
the green color returns.
2- The addition of sodium carbonate to the titration mixture results in
the liberation of CO2, which prevents atmospheric oxidation.
3- If the mixture bumps or it becomes too concentrated during
titration, remove it from the heater, boil 10 ml water in a test tube
and add it to the reaction mixture. Heats the mixture until it boils
again and continues the titration.
4- The tip of the burette must be over the mouth of the flask while
mixture is titrated.

23
Calculation:
The concentration of the sugar in each tube can be calculated from the
following sugar equivalent equation
M * V = M′ * V′
The equivalents for a number of sugars are given as follow, but they only
applied if the above conditions are strictly adhered to.
25 ml of Benedict’s reagent is equivalent to 50 mg of glucose
53 mg of fructose
68 mg of lactose
74 mg of maltose
49 mg of hydrolyzed sugar

Since we used 5ml of Benedict reagent which is equivalent to 9.8 mg of

hydrolyzed sugar
5ml benedict = 9.8 mg
5ml benedict = Titer no. ml
9.8 mg of sugar = Titer no. ml

X mg of sugar = 1 ml
X mg of sugar/ ml = 1 *9.8 /titer No. *dilution factor
Dilution factor= Final volume / Initial volume

References:

1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 1978

2- Harvey,R and Champe,P. Lippincott biochemistry, london.2005

24
Results Sheet

Fill the table below and plot a relationship between the substrate and
product concentration.

[S] Titer number [P]

Tube no.
mM ml mg/ml
1
2
3
4
5

25
Results Sheet

26
 Experiment 7: Estimation of lipase activity

Lipase is a pancreatic enzyme secreted into the small intestine. It

catalyses the hydrolysis of triacylglycerols to free fatty acids and glycerol
as follow:

The release of fatty acids in the solution will cause decrease in the pH and
the rate of the reaction may be followed by:
1- Noting the change of pH with time.
2- Titration the liberated free fatty acids with standard alkali using a
suitable indicator
3- By continues titration using an automatic apparatus, (pH-state)
which keeps the pH constant and at the same time plots a curve of
titer number against time.
Method 2 has been adapted foe this experiment. The liberated free fatty
acids at different enzyme concentrations will be titrated with 0.05 N
NaOH. Since we are using oils as substrates CaCl2 is used as emulsifying
agent for two reasons:
1- to increase the surface area
2- To decrease the surface tension, thus the oil drop is effetely
attacked with the enzyme.

27
Materials:

1- Lipase (1g%)
2- Chloroform (10%)
3- Fresh oil as the substrate
4- Calcium chloride
5- Sodium hydroxide 0.05 N

Procedure
Prepare 6 tubs which contain the following:
Tube 1 2 3 4 5 B
Oil
Substrate 2 2 2 2 2 2
(ml)
CaCl2 1 1 1 1 1 1
Mix well
D.W 8 6 4 2 0 10
Lipase
2 4 6 8 10 -
(ml)
Incubate in a water bath 37C˚
Enzyme
concentration 0.02 0.04 0.06 0.08 0.1 -
(µg)

Titrate the liberated fatty acids with NaOH noting the time of the titration
should not exceed 10 min.
References:
1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 1978
2- Harvey,R and Champe,P. Lippincott biochemistry, london.2005

3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New york. 2002

28
Results Sheet

Draw a graph of enzyme concentration against ml of NaOH has taken. Is

your curve hyperbolic or liner, comment?

Enzyme concentration ml of NaOH (titer no.)

29
Experiment 8: Indirect estimation of lactate dehydrogenase
Lactic acid produced during anaerobic glycolysis can be converted to
pyruvic acid with the aid of the enzyme lactate dehydrogenase when
oxygen becomes available. The hydrogen acceptor NAD+ accepts the
hydrogen atoms from the lactic acid and the pyruvic acid molecule
results. Part of the produced pyruvic acid enters the citric acid cycle after
being converted to acetyl CoA. The remainder of the pyruvic acid is
converted into glycogen.

Lactate Acetyl CoA Citric acid cycle

dehydrogenase
CH3CHOHCOOH CH3COCOOH
Glycogen
Pyruvic acid

In this experiment, yeast will be used as a source of lactate

dehydrogenase. The reaction will be followed by allowing methylene
blue dye to function in place of the natural hydrogen acceptor NAD+. As
methylene blue is reduced it becomes colorless.

Lactate dehydrogenase
CH3CHOHCOOH CH3COCOOH

Methylene Blue Methylene Blue

(blue) (colorless)

30
Materials:
 yeast suspension
 5% sodium lactate solution
 0.1% methylene blue
 Water bath 37˚C
 Boiling water bath

Procedure:
- Label three clean test tubes as a, b and c as followed
- Make sure that you shake the bottle of yeast suspension before
removing your sample
Test tube A B C
2ml
Yeast suspension
Yeast Pre heated for 10 min
2ml 2ml
suspension in boiling water bath
and cooled to 37˚C
before being used
Sodium lactate 10 drops 10 drops
Methylene blue 1 drop 1 drop 1 drop

Continue to add methylene blue drop wise ( mixing after each drop)
until each solution becomes a uniform light blue in color

Mix and place in water bath 37˚C

Observe the tubes after 10 min. Note any color changes and record
your observations

References:
1- Plummer, D. An introduction to practical biochemistry. McGraw-HILL, london. 1978

2- Harvey,R and Champe,P. Lippincott biochemistry, london.2005

3- Boyer, R. Concepts in biochemistry. John Wiley and sons, New york. 2002

31
Results Sheet

Experiment Observation Comment

32
 Experiment 9: Detection of enzymes
Pepsin:
 It is protease found in the digestive system of many vertebrates.
 The pancreas secretes pepsinogen (proenzyme).
 Activated when chief cells in the stomach release it into HCL
which activates it.
 It degrades food proteins into peptides which can be readily
absorbed by the intestine.
 Optimum ph =5

Detection of pepsin:
W.B (37-40°C)
1 ml milk + 1 ml buffer soln. (pH 5) + 0.5 ml pepsin Coagulation occur
(10-20 min)

Lipase
 It is a pancreatic enzyme secreted into the small intestine.
 Catalyses the hydrolysis of triacylglycerols to free fatty acids and
glycerol.
 Optimum ph =8

Detection of lipase:

1 ml milk + 2 drops ph.ph + drops of NaOH (0.1 N) till pink color appear
W.B (37-40°C)
+ 0.5 ml lipase Turns colorless
(10-20 min)

Amylase
 glycosidase that attack starch randomly. Inactivated by the acidity
of the stomach.
 Optimum ph = 7

33
Detection of amylase:
W.B (37-40°C) for 30min
1 ml starch + 1 ml amylase Take 1 ml of the
solution every 5 min, and test the presence of starch by iodine solution.

Urease
 Urease is found in bacteria, yeast, and several higher plants.
 catalyzes the hydrolysis of urea into carbon dioxide and ammonia
urease
 (NH2)2CO + H2O ------------→ CO2 + 2NH3
 Optimum ph = 7.4

Detection of Urease:

2.5 ml urea + drops Na2CO3 + drops phenol red (Red color), then add

acetic acid drop by drop until the color change to (Yellow ) + filter paper
W.B (37-40°C)
soaked in urease red color returns
(10-20 min)

34
Results Sheet

Experiment Observation Comment

35
Experiment 10: Detection of Enzyme mixture

36
Results Sheet

Unknown 1:
Contains…………………………………………………………………

Experiment Observation Comment

37
Results Sheet

Unknown 2:
Contains…………………………………………………………………

Experiment Observation Comment

38
Results Sheet

Unknown 3:
Contains…………………………………………………………………

Experiment Observation Comment

39
Results Sheet

Unknown 4:
Contains…………………………………………………………………

Experiment Observation Comment

40