NONG LAM UNIVERSITY - HO CHI MINH CITY

FACULTY OF CHEMICAL ENGINEERING AND FOOD TECHNOLOGY

ENZYME TECHNOLOGY
LABORATORY SESSION

DR. NGUYEN MINH XUAN HONG

2022
Laboratory Safety

Safety is of paramount importance in this (or any) laboratory. Additional Safety Notes
are included in each of the experiments in this manual. In addition, laboratory
instructor will give specific guidelines on the safe conduct of each experiment at the
beginning of the laboratory period. Non-attendance during this safety briefing can
result in students being barred from participation in the laboratory experiment.

The following comments on the general rules as listed may help to clarify any
questions that arise. Please inquire of your instructor or the lab manager if there are
further questions.

1. Working without supervision is forbidden. A student should never stay in the

laboratory alone or work without a instructor present.

2. Performing unauthorized experiments or any experiment at unauthorized times

is forbidden. Unanticipated results to an unauthorized or altered experiment can
be quite hazardous. Materials should never be taken from the laboratory.
Horseplay and pranks are never acceptable in the laboratory.

3. Approved safety eyewear must be worn when needed. Consult your instructor
about the policy for wearing contact lenses in your laboratory. Students may
wear contact lenses, but should inform the instructor, so that should an accident
occur, the instructor is aware that the lenses are in your eyes. Certain solvents
in use during some experiments can have adverse effects on soft contacts and
students should take appropriate precautions following those labs.

4. Loose hair and clothing must be restrained. Hair or clothing that can get caught
or otherwise be a hazard in lab should be pulled back.

5. Appropriate attire must be worn in lab, including shoes that cover the entire
foot, and clothing which covers the legs and torso.

6. Eating, drinking, smoking, chewing tobacco and applying cosmetics are strictly
forbidden in the laboratory.

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7. Items such as book bags, backpacks, purses and coats should be put out of way
off the floor and lab bench on one of the hooks provided.

8. Cell phones should be turned off before entering the laboratory.

9. All accidents and breakage must be reported to an instructor. Any accident

requires attention to clean up. Alert your neighbors immediately if there is a
spill as to its nature and extent before you leave the area.

10. Pipetting by mouth suction is forbidden. Pipet bulbs and/or dial-up pipetters
will be provided when necessary.

11. When needed, gloves must be worn. Gloves should be of a material and
thickness appropriate for the reagents being used. However, gloves provide
only a temporary layer of protection against chemicals on your skin and may
be permeable to some chemical reagents, without visible deterioration. If your
gloves come in contact with a chemical reagent, remove them, wash your
hands, and get a new pair immediately.

12. Read labels and dispose of any waste in an appropriate waste container. Ask
questions if you are not sure what to do.

13. All materials used in the laboratory have a Material Safety Data Sheet (MSDS)
on file. If you wish to see one at any time, inquire the lab manager.

14. Clean up in the laboratory is essential. Make sure you wash and put away all
equipment before you leave the laboratory.

15. Hands must be washed just before leaving the laboratory.

16. Inform your instructor at the beginning of the term if you have any special
medical conditions that may need attention during the laboratory, such as
known allergies, seizures.

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 Lab 1: ENZYME KINETICS

Solutions

Sodium Phosphate buffers (0.02 M): pH4, pH6 and pH8

For pH 5 and above, use NaH2PO4 and Na2HPO4.

For pH 4 and below, use NaH2PO4 acidified with phosphoric acid.

Starch solution (10g/L = 1%):

Make a starch solution of 10g/L soluble starch in warm sodium phosphate buffer,
pH 7.0. Heat starch solution to boiling (or microwave for 5 minutes). Starch
should go fully into solution after heating. Solution will appear cloudy.

α-Amylase solution:

Dilute amylase solution 100-fold in phosphate buffer. Keep enzyme solution on

ice during experiments. Store enzyme and enzyme stock solution at 4ºC (in the
refrigerator).

Lugol solution

10 g KI + 5 g I2 in water to 100 mL total → Iodine content = 130 mg/mL.

Fehling solution
Saccharose solution 1%
NaCl solution 1%
CuSO4 solution 1%

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1. Specificity of enzyme

Prepare 3 test tubes. Add 0.5 mL of 1% enzyme amylase into each tube. Then add 1mL
of 1% starch into tube 1, 1mL of 1% saccharose into tube 2 and 1mL of distilled water
into tube 3. Keep 3 test tubes into water bath at 37oC in 5 minutes.

Test Fehling reaction: Mix 2 mL of Fehling A and 2 mL of Fehling B in 1 test tube. Put
1 mL of the mixed Fehling solution into each above test tube. Put these test tubes in hot
water for 1-3 minutes. Observe and compare the results.

Tube 1:

Tube 2:

Tube 3:

2. Effect of enzyme concentration on enzyme activity

Prepare 5 tubes containing enzyme amylase and starch as following:

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

Distilled water 1 mL 0.9 mL 0.8 mL 0.5 mL 0 mL

1% Enzyme 0 mL 0.1 mL 0.2 mL 0.5 mL 1 mL

1% Starch 1 mL 1 mL 1 mL 1 mL 1 mL

 [Enzyme] 0% 0.05% 0.1% 0.25% 0.5%

 Reaction
time (min)

Check the reaction with Lugol solution after each 30 seconds until all reactions have
completed. Record the time.

Plot the relationship between enzyme concentration and reaction time.

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3. Effect of substrate concentration on enzyme activity

Prepare 5 tubes containing starch and enzyme amylase as following:

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

Distilled water 1.8 mL 1.6 mL 1.3 mL 0.8 mL 0.3 mL

1% Starch 0 mL 0.2 mL 0.5 mL 1.0 mL 1.5 mL

1% Enzyme 0.2 mL 0.2 mL 0.2 mL 0.2 mL 0.2 mL

 [Starch] 0% 0.1% 0.25% 0.5% 0.75%

 Reaction time
(min)

Check the reaction with Lugol solution after each 30 seconds until all reactions have
completed. Record the time.

Plot the relationship between starch concentration and reaction time.

4. Effect of temperature on enzyme activity

Equilibrate 2 tubes containing 1 mL of 1% starch solution, 1 tubes containing 0.1 mL of

1% enzyme solution at each assay temperature (0, 30, 100°C).

Quickly add the contents of one starch tube to enzyme tubes (keeping the solutions at
the desired assay temperature).

Be sure to run a control set of three starch tubes with 0.1 mL of buffer instead of enzyme
solution for each temperature.

Check the reaction with Lugol after each 30 seconds until all reactions have completed.
Record the time. Plot the relationship between temperature and reaction time.

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5. Effect of pH on enzyme activity

Prepare 3 tubes containing 0.5 mL of 1% starch solution. Add 2 mL of buffer at different

pH (4, 6, 8) to each tube and shake well. Then add 0.1 mL of 1% enzyme solution to
each tube.

Be sure to run a control set of three starch tubes with 0.1 mL of buffer instead of enzyme
solution for each pH.

Check the reaction with Lugol solution after each 30 seconds until all reactions have
completed. Record the time. Plot the relationship between pH and reaction time.

6. Effect of Inhibitor and Activator on enzyme activity

Prepare 3 tubes as following:

Tube 1: 0.5 mL of 1% amylase solution and 0.5 mL of 1% NaCl

Tube 2: 0.5 mL of 1% amylase solution and 0.5 mL of 1% CuSO4

Tube 3: 0.5 mL of 1% amylase solution and 0.5 mL of distilled water

Add 1mL of 1% starch solution to each tube. Keep it at room temperature for 1-3
minutes. Then check the reaction with Lugol solution. Observe and compare the results.

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 Lab 2: DETERMINATION OF ENZYME
AMYLASE ACTIVITY
1. Background
Enzyme α-amylase breaks down starch (and glycogen, since the two are
chemically very similar) by addition of water between glucose units (hydrolysis). The
end products of the amylase reaction are shorter polymers of glucose along with the
disaccharide maltose, while glucoamylase liberates single glucose sugars. Amylase
occurs in mammalian saliva and small intestines and is found in the intestinal tract of
most animals. It is also common in plants, where it degrades stored starch and in some
bacteria and fungi that attack plants. Enzyme α-amylase is widely used in many
industries such as pharmaceuticals, baking, detergents, sewage treatment, natural
sweeteners, and animal feeds (Guzman-Maldonado et al. 1995). α-Amylase catalyzes
hydrolysis of α-1, 4-glucosidic linkage of α-1, 4-glucans, such as starch, glycogen, and
dextrins. Starch hydrolysis by amylase to produce maltodextrins is one of the major
industrial enzyme-catalyzed reactions.
This assay is based on the method of Heinkel (1956) with some modifications.
Buffer and starch are mixed in a test tube, and the reaction is initiated by addition of
enzyme. After incubation, sulfosalisilic acid is added to the tube to terminate the
reaction. Any starch remaining in the tube turns blue, with the intensity of the blue color
being proportional to the amount of starch present. The absorbance of this solution is
measured using a spectrophotometer at a wavelength of 560 nm. The greater the change
in color and absorbance between a control sample of starch (without enzyme) and the
reaction mixture, the greater the amount of starch degraded by the enzyme; thus, the
greater the activity of the enzyme being measured.

The commercially available enzymes for this laboratory are α-Amylase

(Termamyl).

Activity unit of enzyme amylase is the amount of amylase to hydrolyse 1 mg

starch after 30 minutes at 30oC.

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2. Reagents
Sodium Phosphate buffer 0.05 M, pH6

1% starch solution (10g/L)

1% starch solution in Sodium Phosphate buffer 0.05 M, pH6

1% α-Amylase solution

Iodine Solution: dissolve 1g Iodine and 2g KI in 300mL distilled water. Dilute

this solution 150 times when using.

0.1% NaCl solution

20% sulfosalisilic acid solution

3. Procedure

3.1 Standard curve: Corellate A590 to starch concentration

Prepare 6 dilutions of the starch solution. Label tubes D1-D6 and dilute starch
solution with water as described in following table.

D1 D2 D3 D4 D5 D6

1% starch 0mL 2mL 4mL 6mL 8mL 10mL

Distilled
10mL 8mL 6mL 4mL 2mL 0mL
water

[starch] 0mg/mL 2mg/mL 4mg/mL 6mg/mL 8mg/mL 10mg/mL

Prepare another set of 6 tubes. Put 0.1mL from each of the above dilutions. Add
0.1mL of 0.1% NaCl, 0.2mL of phosphate buffer, 0.1mL distilled water, 0.5mL of 20%
sulfosalisilic acid and shake well. Add 9mL of 150-time diluted Iodine solution.
Measure the absorbance (A560) of each of the tubes and record the values. Plot the
standard curve between A560 and mg of starch.

D1 D2 D3 D4 D5 D6

mg starch 0 0.2 0.4 0.6 0.8 1.0

OD560 0.064 0.275 0.491 0.751 0.987 1.262

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3.2 Enzyme activity

Put 1 mL of 1% starch solution, 1 mL of 0.1% NaCl and 2mL of phosphate buffer

into 2 test tube. Shake well and keep at 30oC in 30 minutes. Add 1mL of enzyme at 30oC
to tube 1 and 1mL of distilled water to tube 2 and keep at 30oC in another 30 minutes.
Add 5mL of 20% sulfosalisilic acid to each tube and shake well to stop the reaction.
Filter the mixtures to remove any precipitate.

Take 1mL of each filtrate into 2 new tubes. Add 9mL of 150-time diluted Iodine
solution. Measure the absorbance (A560) of each of the tubes and record the values.
Subtract the absorbance readings for the enzyme reaction tubes from the control reading.
Correlate the change in absorbent (A560) with the standard curve to determine how
much starch was degraded in the alloted time. Larger A560 represents higher levels of
activity.

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 Lab 3: ENZYME PROTEASE

1. Background
Pepsin is the principal proteolytic enzyme of vertebrate gastric juice. Its inactive
precursor form, pepsinogen, is produced in stomach mucosa. Pepsin was first recognized
in 1836 by the German physiologist Theodor Schwann. In 1930 it was crystallized and
its protein nature established by John H. Northrop of the Rockefeller Institute for
Medical Research.

Pepsin has broad specificity with a preference for peptides containing linkages
with aromatic or carboxylic L-amino acids. It preferentially cleaves C-terminal to Phe
and Leu and to a lesser extent Glu linkages. The enzyme does not cleave at Val, Ala, or
Gly.

Pepsin is a monomeric, two domain, mainly beta protein with a high percentage
of acidic residues. Porcine pepsin has 4 basic residues, and 42 acidic residues and is O-
phosphorylated at S68 (Tang et al. 1973). For the protein to be active, one of the two
aspartate residues in the catalytic site has to be protonated, and the other deprotonated.
This occurs between pH 1 and 5, and above pH 7 pepsin is irreversibly denatured.

Pepsin is most active in acidic environments between 37°C and 42°C.

Accordingly, its primary site of synthesis and activity is the stomach (pH 1.5 to 2).
Pepsin exhibits maximal activity at pH 2.0 and is inactive at pH 6.5 and above, however
pepsin is not fully denatured or irreversibly inactivated until pH 8.0. Therefore pepsin
in solution of up to pH 8.0 can be reactivated upon re-acidification. The stability of
pepsin at high pH has significant implications on disease attributed to laryngopharyngeal
reflux. Pepsin remains in the larynx following a gastric reflux event. At the mean pH of
the laryngopharynx (pH = 6.8) pepsin would be inactive but could be reactivated upon
subsequent acid reflux events resulting in damage to local tissues.

Pepsin is prepared commercially from swine stomachs. Crude pepsin is used in

the leather industry to remove hair and residual tissue from animal hides prior to their
being tanned. It is also used in the recovery of silver from discarded photographic films
by digesting the gelatin layer that holds the silver compound.
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2. Extraction of pepsin

Always keep the environment at 0-4oC during the extraction of enzyme.

Turn the fresh stomach reversely and gently wash leftover food. Remove fat and
weight the stomach.

Separate the mucous membrane on the stomach: The thicker white part and the
thinner brown part which contain more enzymes.

Cut the membrane as small as possible. Take 10g of the membrane to soak in
0.8% HCl with the ratio of 1:2 weigh. Adjust the pH to 1.5-2. Keep the sample at 40oC
in 24 hours for hydrolyzation.

Filter the mixture to remove all insoluble. Measure the volum of the filtrate.

3. Determine the activity of crude pepsin by modified method of Anson (1938)

3.1 Principle

Pepsin hydrolyzed casein in acidic condition to produce peptides which are not
precipitated by trichloro acetic acid (TCA). Tyrosin and tryptophan in casein is
determined by color reaction with Folin reagent. Measure the absorbance of the solution
to determine the enzyme activity.

3.2 Reagents

• 2% casein solution

• 5% trichloro acetic acid solution

• 0.5N NaOH

• Folin reagent diluted 3 times with distilled water

• 1m tyrosin standard solution (181,19 mg Tyrosin in 1L of 0.2N HCl)

• 0.2N HCl

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3.3 Standard curve of tyrosin

Label 6 test tubes and put the reagents as directed in the following table:

1 2 3 4 5 6
1m tyrosin solution (ml) 0 0.2 0.4 0.6 0.8 1
0.2N HCl (ml) 1 0.8 0.6 0.4 0.2 0
0.5N NaOH (ml) 2 2 2 2 2 2
Folin (ml) 0.6 0.6 0.6 0.6 0.6 0.6

Let the solution settled for 15 minutes. Then measure the A660nm of these solutions.
Draw the standard curve between A660 and m tyrosin.

1 2 3 4 5 6

m tyrosin 0 0.2 0.4 0.6 0.8 1

OD660 0.021 0.312 0.497 0.687 0.83 1.019

1 Anson unit is the quantity of enzyme to hydrolyze substrate in 1 minute to make

product equivalent to 1 mol tyrosin.

3.4 Procedure

Label 2 test tubes as “A” (enzyme test) and one test tube as “B” (control). Put 5mL of
2% casein in 3 test tubes. Put 1mL of crude extract of pepsin into each tube A, incubate
at room temperature for 10 minutes, then add 10mL of 5% TCA. Tube B is added with
10mL of 5% TCA first, incubate 10 minutes, then add 1mL of crude enzyme extract.

Incubate all tubes for 30 minutes, and then filter. Take 1mL filtrate from each solution
to add with 2mL of 0.5N NaOH and 0.6mL of Folin reagent. Incubate for 15 minutes,
then measure the A660. Correlate the change in absorbent (OD) with the standard
curve to determine the quantity of tyrosin, then determine the activity unit of pepsin.

OD = (ODA1 + ODA2) /2 `– ODB

mol Tyr x V
Protease activity / 1 mL enzyme =
t

V : volume of the mixture (=16 mL)

t : time for hydrolyzation (= 10 minutes)

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4. Effect of enzyme bromeline on meat tenderizing

Bromelin is a natural plant-derived proteolytic enzyme found in members of the

Bromeliaceae family, typified by Pineapple. Because of their catalytic activity, cysteine
proteinases are commonly used in the food and pharmaceutical industries and in
diagnostic laboratories.

Pineapple was removed the hard shell and cut into small pieces, then the juice is
extracted using a juicer or filter cloth.

Meat is cut into long thin pieces which their muscle can be seen clearly. Divide
the meat into 4 parts and put into 4 dishes:

Dish 1: control, no heat-treated

Dish 2: add pineapple juice

Dish 3: control, heat-treated (cook for 5 minutes)

Dish 4: add pineapple juice, incubate at room temperature in 3 hours, then

cook for 5 minutes

Observe and compare the structure of the meat in 4 dishes.

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 Lab 4: ENZYME PECTINASE

1. Background
Pectic substances are complex high molecular mass glycosidic macromolecules
found in higher plants. They are present in the primary cell wall and are the major
components of the middle lamellae, a thin extracellular adhesive layer formed between
the walls of adjacent young cells. In unripe fruit, pectin is found as a water insoluble
pectic substance, the protopectin, bounded to cellulose microfibrils conferring rigidity
on cell walls. During ripening the fruit enzymes alter the pectin structure by breaking
the pectin backbone or side chains, resulting in a more soluble molecule.

Pectinases are a big group of enzymes that break down pectic polysaccharides of
plant tissues into simpler molecules like galacturonic acids. It has long been used to
increase yields and clarity of fruit juices. Pectinases are an enzyme group that catalyzes
pectic substance degradation through depolymerization (hydrolases and lyases) and
deesterification (esterases) reactions.

2. Determination of pectinase activity using color-metric method

2.1 Principle

The activity of pectinase was assayed by measuring the monomeric galacturonic acids
released by the enzyme by catalysing the pectin degradation. The results were expressed
as specific activity units.

One unit of enzyme activity was defined as the amount of enzyme that hydrolyse 1g
pectin to galacturonic acids after 60 minutes at standard condition (30oC, pH 3.9-4.1).

2.2. Reagents

• 1% pectin solution
• 15% ZnSO4 solution
• 1% enzyme pectinase solution
• 0.2% Anthrone solution in sulphuric acid

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2.3 Procedure

Mix 10 mL of 1% pectin solution with 5 mL of 1% enzyme pectinase solution in

a test tube and keep at 30oC for 60 minutes (pH of pectin solution = 3.9-4.1). A control
sample is also done with water instead of enzyme. After that, 2 mL of 15% ZnSO4
solution is added and the mixture is filtered using filter paper. The filtrate is diluted 5
times. Then, 5 mL of Anthrone solution is mixed vigorously with 2.5 mL of diluted
filtrate in 10 minutes. Then it is kept in water bath at 70oC in 12 minutes. After that, it
is cooled down to room temperature and has its OD measured at A584.

 OD = ODT – ODCT

0.34 x  OD – 0.0104
Pectinase activity =
m

ODT : A584 of the test sample

ODCT : A584 of the control sample
m : amount of enzyme used (g or mL)

3. Application of pectinase in clarification of apple juice

Weight an apple and cut into pieces. Using a juicer to extract the juice from the apple.

Dilute 20 mL of the juice with water to make 100 mL solution of apple juice.

Label 4 test tubes and add solution as following:

Tube 1 2 3 4

Apple juice 8 mL 8 mL 8 mL 8 mL

Pectinase 0 mL 0.2 mL 0.5 mL 1.0 mL

Water 2 mL 1.8 mL 1.5 mL 1.0 mL

OD584

Keep all tubes at 45-50oC in 30 minutes. Ater that, the clarified juice of the upper
part in each tube is separated to measure the A584. Plot the A584` with enzyme
concentration. Explain and discuss the results.

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Lab Reports

The lab reports should contain abstract, background, materials and methods, results, and
discussion sections. All sections should be written with complete sentences in paragraph
form. Below are descriptions of what should be included in each section. The
contribution of each section to the grade for an individual lab report is given in
parentheses beside the section name.

Abstract (20%) – 4-6 sentences summarizing the goal of the experiment, the techniques
used, the findings and their relevance.

Background (10%) – 1-2 paragraphs describing why you are doing the particular
experiment and providing the context to understand the results. Write this as if you are
explaining the experiment to an educated colleague whom has never been exposed to
that particular procedure.

Materials and Methods (10%) – 1-2 paragraphs describing the techniques and
procedures used in the particular experiment. Use complete sentences, and write out
equations as necessary. If an equation is included, please give an example of a
calculation using the equation where you use real numbers.

Results (30%) – 1-2 paragraphs describing your observations. Any tables and figures
reported in this section must include legends that describe the information contained in
the table or figure and how you interpret the data reported therein.

Discussion (30%) – 2-3 paragraphs describing your interpretations of the results of the
experiment. Explain what the results mean and what you conclude from them. Be sure
to explain your reasoning for drawing your particular conclusions.

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