VIETNAM NATIONAL UNIVERSITY HO CHI MINH CITY – VNU-HCMC

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY

FALCUTY OF CHEMICAL ENGINEERING

LABORATORY OF

FOOD CHEMISTRY AND BIOCHEMISTRY

REPORT EXPERIMENT 5: ENZYME
Experiment 5.1 Activity of amylase preparation and digestion of protein
Experiment 5.2 Comparison of invertase hydrolysis and acidic hydrolysis for
saccharose

CLASS: CC01 – GROUP: 2 – SEMESTER: 212

INSTRUCTOR: Prof. Dr. Võ Đình Lệ Tâm
GROUP’S MEMBERS:

No. Name Student ID

1 Vũ Trọng Thức 2052274

2 Nguyễn Minh Nhật 2053298

3 Phạm Thị Ngọc Anh 1952568

Feburary 20

1
 EXPERIMENT 5.1: ACTIVITY OF AMYLASE PREPARATION AND
DIGESTION OF PROTEIN

1. Determine of amylase preparation activity by Wohlgemuth’s procedure

1.1 Introduction

The principle of Wohlgemuth's method is the extraction of a weighed portion of

starch with 1 per cent, NaCl solution and the determination of the amylolytic activity of
gradually decreasing volumes of this centrifugated extract, by means of a series of tubes.

1.2 Principle

Amylase is an enzyme preparation which can hydrolyze starch into dextrin, maltose,
and glucose.

Wohlgemuth method is used to determine amylase acitivity by finding the smallest

amount of enzyme in order to hydrolyze a known amount of starch at a defined condition
until the mixture no longer reacts whith Lugol reagent.

One Wohlgemuth activity unit is a needed amount of enzyme in order to hydrolyze

1mg of starch in 30 minutes at 37ºC with Cl ̄ as an activator.

1.3 Apparatus

- Փ 12 test tube – 10 tubes + test tube rack (Table 1)

- Փ 18 test tube – 07 tubes + test tube rack (Table 2)

- Bowl, spoon (for weighing) + 100ml volumetric flask (for soaking malt)

- Funnel (for filtering) + 2 filter papers + 100ml beakers (for filtering malt extract)

- Pipette: 1ml pipette: 2 + 5ml pipette: 2 + 10ml pipette: 2

- 100ml beaker: 2

- Stirring rod + petri dics (tesing samle with Lugo reagent) – 2 kits

- Other tools: rubber buld + dispensing bottle + cleansing paper.

1.4 Materials and Chemicals

- Maltaz powder (amylase preparation): 10g/group

- 0.5% starch solution (completedly gelatinized)

2
- 1% starch solution (completely gelatinized)

- 0.5% NaCl solution

- 10% H2SO4 solution

- Lugol reagent: 0.3% of I2 in 3% KI solution (0.3% I2/ 3% KI solution)

- 0.5% glucose solution

- Acetate buffer: pH 3.0 - 4.0 - 5.0 – 6.0 – 7.0

- Distilled water

1.6 Procedure

Preparartion of Amylase extract

- Weigh 10g of Maltaz powder, put into 100ml volumetric flask, fill 100ml line
with distilled water, shake well.
- Soak the Maltaz powdwe in 60 minutes, shake occasionally.
- Filter the mixture through a double-layer filter paper (2 filter papers) to collect a
transparent supernatant (should re-filter 5-10ml of the supernatant which is
collected at the frist stage in order to recover a completely clear supernatant).

Examine amylase activity

- Take 10 test tubes of Փ12, number them from 1 to 10.

- Put in each test tube 1ml of 0.5% NaCl solution.
- Put 1ml of amylase extract in the first test tube [1], shake well. Then, take 1ml
from [1], put into the second test tube and shake well. Repeat for the following
test tubes until the 10th test tube then discard 1ml of solution from it.
- Next, put in each test tube 1ml of 0.5% starch solution, shake well, then incubate
at 37ºC, shake occasionally for hydrolyzing all starch gains on the tube wall. After
30 minutes, put in each test tube ml of 10% H2SO4 solution (to deactivate the
enzyme) and 2 drops of Lugol reagent, shake well, observe the color change in
each test tube.
- Mark the tube having the lowest enzyme amount at which the starch was
hydrolyzed completely, which means it has the light yellow color, the tube after

3
 it is the tube has the red color, red-purple color… (the starch was hydrolyzed
incompletely)
- Table 1 illstrate test result, name color of the tube in the table (blue [B], purple
[P], brown [B], red [R], orange [O], yellow [Y]).

1.6 Calculation

Amount of enzyme in the test tube [1] (n)

.
n=

Where

V1: Volume of amylase extract in the tube [1], ml

V2: Volume of amylase extract, ml

m: Malt mass use extract amylase, mg

One unit of Wohlgemuth (W)

W=
.

Where

F: Dilution factor chosen from the Table 1

Number of activity unit (Wohlgemuth) in 1ml of amylase extract (N)

Nw =
.

1.7 Result and report

Table 1. Result of the experiment of determination of amylase acitivity

Tube no. 1 2 3 4 5 6 7 8 9 10

Dilution 2 4 8 16 32 64 128 256 512 1024

factor (F)

Enzyme n/2 n/4 n/8 n/16 n/32 n/64 n/128 n/256 n/512 n/1024
amount

4
 Color of
solution

2. Effect of temperature (optional), pH and enzyme amount (optional) on the

activity of amylase preparation

2.1 Introduction

Amylases, which are starch-degrading enzymes, are grouped into endoamylases,

exoamylases, debranching enzymes, and transferases. α-Amylase is an endoamylase that
catalyzes the hydrolysis of internal α-1,4-glycosidic linkages into glucose, maltose, and
maltotriose units. There are three types of amylases regarding the temperature and pH
condition. Regular amylases may be applied at pH 5.5–7.0 and 25–55°C. Medium-
temperature amylases can be used above 50°C, while high-temperature amylases can be
used at boil and in a padding process.

2.2 Principle

Enzyme is a protein-nature bio-catalyst, therefore, its activity is restrained by the

reaction condition such as temperature, pH, and enzyme amount.

Amylase is an enzyme which can hydrolyze starch into dextrin, maltose, and glucose.
In the digestive tract, alpha-amylase enzyme in saliva can hydrolyze gelatinized starch in
food, and other amylase with finish hydrolysis to generate glucose, which is then absorbed
through intestine wall.

2.3 Apparatus

- Փ 12 test tube – 10 tubes + test tube rack (Table 1)

- Փ 18 test tube – 07 tubes + test tube rack (Table 2)

- Bowl, spoon (for weighing) + 100ml volumetric flask (for soaking malt)

- Funnel (for filtering) + 2 filter papers + 100ml beakers (for filtering malt extract)

- Pipette: 1ml pipette: 2 + 5ml pipette: 2 + 10ml pipette: 2

- 100ml beaker: 2

- Stirring rod + petri dics (testing sample with Lugo reagent) – 2 kits

5
- Other tools: rubber bulb + dispensing bottle + cleansing paper.

1.4 Materials and Chemicals

- Maltaz powder (amylase preparation): 10g/group

- 0.5% starch solution (completely gelatinized)

- 1% starch solution (completely gelatinized)

- 0.5% NaCl solution

- 10% H2SO4 solution

- Lugol reagent: 0.3% of I2 in 3% KI solution (0.3% I2/ 3% KI solution)

- 0.5% glucose solution

- Acetate buffer: pH 3.0 - 4.0 - 5.0 – 6.0 – 7.0

- Distilled water

1.6 Procedure

Preparation of standard color test tube

1. 2ml of 1.0% starch solution + 3 drops Lugol reagent

2. 2ml of 0.5% glucose solution + 3 drops Lugol reagent

Effect of temperature on enzyme activity

- Number 4 test tubes (Փ18) from 1 to 4

- Put in each test tube in order: 5.5ml of pH 6.0 acetate buffer solution + 4ml of
0.5% starch solution (check pH value) + 0.5ml of amylase extract (10ml total).
- Set the test tubes at different temperature for reaction: [1] 20ºC – [2] 30ºC – [3]
50ºC – [4]70ºC.
- Check result: after 15 minutes, take 1 drop of sample from each test tube, drop it
separately on a Petri dish, add 1 drop of Lugol reagent, observe the color,
comment and explain.
- Determine the hydrolysis time: take 1 drop of sample from each test tube after 3
minutes periodically, drop it separately on a Petri dish, add 1 drop of Lugol
reagent, observe the color, continue until the color of the sample droplet is the
same as that of test tube [2]. Note the hydrolysis time.

6
 Note: Should dilute Lugol reagent 10 times, so that the color is not too dark.

Effect of pH on enzyme activity

- Number 4 test tubes (Փ18) from 1 to 4

- Put in each test tube in order: 5.5 ml buffer solution + 4ml of 0.5% starch solution
(check pH value and adjust by 0.2M acetic acid or 0.2M acetate sodium) + 0.5ml
of amylase extract (10ml total).
- Test pH value: [1] 3.5 – [2] 4.0 – [3] 5.0 – [4]6.0 for each tube.
- Check the result: after 15 minutes, take 1 drop of sample from each test tube, drop
it separately on a Petri dish, add 1 drop of Lugol reagent, observe the color,
comment and explain.
- Determine the hydrolysis time: take 1 drop of sample from each test tube after 3
minutes periodically, drop it separately on a Petri dish, add 1 drop of Lugol
reagent, observe the color, continue until the color of the sample droplet is the
same as that of test tube [2], note the hydrolysis time (min).
Note: Should dilute Lugol reagent 10 times, so that the color is not too dark.

Effect of enzyme amount on its activity

- Number 4 test tubes (Փ18) from 1 to 4

- Put in each test tube in order: y ml distilled water (pH 7) + 4ml of 0.5% starch
solution + x ml of amylase extract.
- Enzyme amount: x = 0.24ml – 0.5ml – 0.75ml – 1ml amylase extract
- y = 5.75ml – 5.5ml – 5ml distilled water.
- Check the result: after 15 minutes, take 1 drop of sample from each test tube, drop
it separately on a Petri dish, add 1 drop of Lugol reagent, observe the color,
comment and explain.
- Determine the hydrolysis time: take 1 drop of sample from each test tube after 3
minutes periodically, drop it separately on a Petri dish, add 1 drop of Lugol
reagent, observe the color, continue until the color of the sample droplet is the
same as that of test tube [2], note the hydrolysis time in minute.

Note: Should dilute Lugol reagent 10 times, so that the color is not too dark.

2.6 Result and report

7
 Table 2. Experiment layout

Test tube 1 2 3 4

Standard color test tube

0.5% starch solution, mL 2

0.5% Glucose solution, mL 2

Effect of temperature on enzyme activity

Buffer solution, mL 5.5 5.5 5.5 5.5

0.5% starch solution, mL 4 4 4 4

Amylase extract, mL 0.5 0.5 0.5 0.5

pH 6 6 6 6

Temperature, ℃ 20 30 50 70

Results:

Color

Hydrolysis time (min)

Comment and explain

Effect of pH on enzyme activity

Buffer solution, mL 5.5 5.5 5.5 5.5

0.5% starch solution, mL 4 4 4 4

Amylase extract, mL 0.5 0.5 0.5 0.5

pH 3.0 4.0 5,0 6.0

Temperature, ℃ 30 30 30 30

Results:

Color

8
 Hydrolysis time (min)

Comment and explain

Effect on enzyme amount on its activity

Distilled water, mL 5.75 5.5 5.25 5

0.5% starch solution, mL 4 4 4 4

Amylase extract, mL 0.25 0.5 0.75 1

pH 7 7 7 7

Temperature, ℃ 30 30 30 30

Results:

Color

Hydrolysis time (min)

Comment and explain

3. Digestion of protein

3.1 Introduction

The digestion of protein entails breaking the complex molecule first into peptides,
each having a number of amino acids, and second into individual amino acids. The pepsins
are enzymes that secreted by the stomach in the presence of acid that breaks down proteins
(proteolysis).

3.2 Principle

Pepsin is an enzyme working in high acidic environment of stomach. Pepsin

can hydrolyze protein in food to smaller fragments such as polypeptide and peptide.

3.3 Apparatus

- 5 test tubes (Փ18) + test tube rack

- pH stick paper, stick note, name pen

9
 - Water bath

- Tube shaker (vortex)

3.4 Chemicals

- 0.5% pepsin solution

- 0.1% NaOH solution

- 0.8% HCl solution

- Casein

3.5 Procedure
- Number 5 test tubes (Փ18) from 1 to 5.
- Use sticker or name pen marking 2 points 4cm and 8 cm from the bottom of each
test tube.
 Test tube 1: put distilled water to 4-cm-point and 0.8% HCl solution to 8-cm
point.
 Test tube 2: put 0.5% pepsin solution to 4 cm mark. Put the tube in the water
bath (boiling) for 10 minutes, then cool it quickly by tap water, next add 0.8%
HCl solution to 8 cm mark.
 Test tube 3: put 0.5% pepsin solution to 4cm mark, next add 0.1% NaOH
solution to 8 cm mark.
 Test tube 4: put 0.5% pepsin solution to 4cm mark, next add distilled water to
8 cm mark.
 Test tube 5: put 0.5% pepsin solution to 4cm mark, next add 0.8% HCI
solution to 8 cm mark.
- Number pH indicator papers, dip them in corresponding test tubes, note pH value
of each tube.
- Add to each tube 0.1-0.5g casein, vortex it.
- Incubate all tubes at 37°C (body temperature) in 15 minutes.
- Observe the turbidity of each tube, explain.

3.6 Result and report

10
 Tube number pH value Turbidity and
explanation

4. Question

1. Present factors affecting enzyme activity.

Factors that affecting enzyme activity are temperature, pH and the enzyme amount.

2. Present principle of Wohlgemuth method.

Wohlgemuth method is used to determine amylase activity by finding the smallest amount
of enzyme in order to hydrolyze a known amount of starch at a defined condition until the
mixture no longer reacts with Lugol reagent.

One Wohlgemuth activity unit is a needed amount of enzyme in order to hydrolyze 1mg of
starch in 30 minutes at 37ºC with Cl ̄ as an activator.

3. Classify enzyme, give some examples of common enzymes, digestive

enzymes.

 Enzyme classification:

Table 1: Enzymes and its biochemical property

Type Biochemical Property

Oxidoreductases Catalyzes the oxidation reaction where the electrons tend to travel from
one form of a molecule to the other.

Transferases Help in the transportation of the functional group among acceptors and
donor molecules.

11
Hydrolases Hydrolytic enzymes that catalyze the hydrolysis reaction by adding water
to cleave the bond and hydrolyze it.

Lyases Adds water, carbon dioxide or ammonia across double bonds or eliminate
these to create double bonds.

Isomerases Catalyze the structural shifts present in a molecule, thus causing the change
in the shape of the molecule.

Ligases Known to charge the catalysis of a ligation process.

 Example of some specific enzymes, digestive enzymes:

- Amylase: helps change starches into sugars. Amylase is commonly found in saliva.

- Lipases: a group of enzymes that help digest fats in the gut.

- Lactase: found in the small intestine, breaks lactose, the sugar in milk, into glucose
and galactose.

- Maltase: Commonly found in food such as potatoes, pasta and beer.

- Trypsin (Protease): found in the small intestine, breaks proteins down into amino
acids.

4. Present enzyme specificity.

One of the properties of enzymes that makes them so important as diagnostic and
research tools is the specificity they exhibit relative to the reactions they catalyze. A few
enzymes exhibit absolute specificity; that is, they will catalyze only one particular
reaction. Other enzymes will be specific for a particular type of chemical bond or
functional group. In general, there are four distinct types of specificity:

 Absolute specificity - the enzyme will catalyze only one reaction.

 Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.

 Linkage specificity - the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.

 Stereochemical specificity - the enzyme will act on a particular steric or optical

isomer.
12
Though enzymes exhibit great degrees of specificity, cofactors may serve many
apoenzymes. For example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for a
great number of dehydrogenase reactions in which it acts as a hydrogen acceptor. Among
them are the alcohol dehydrogenase, malate dehydrogenase and lactate
dehydrogenase reactions.

5. What is the optimum temperature and pH of trypsin?

The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively.

6. Present the characteristic of pepsin.

Pepsin is an enzyme working in high acidic environment of stomach. Pepsin can hydrolyze
protein in food to smaller fragments such as polypeptide and peptide. Moreover, pepsin is
also a stomach enzyme that serves to digest protein that found in ingested food.
7. What is the application of enzyme in food processing?

Several enzymes currently used in food processing in starch hydrolysis, are actually
produced by recombinant microorganisms. Besides, there are various microbial enzymes
that have been used in food industry.

Table 2: Application of microbial enzymes in food industry

Microbial enzyme Application

α-Amylase Baking, brewing, starch liquefaction

Bread quality improvement
Rice cakes
Clarification of fruit juice

Glucoamylase Beer production

Bread quality improvement

High glucose

High fructose syrups (HFS)

13
 Protease Brewing
Meat tenderization
Coagulation of milk
Bread quality improvement

Lactase (β-galactosidase) Lactose intolerance reduction in people

Prebiotic food ingredients

Lipase Cheese flavour development

Cheddar cheese production

Pectinase Clarification of fruit juice

Glucose oxidase Food shelf-life improvement

Food flavour improvement

5. References

[1] Ciborowski, P., & Silberring, J. (2016). Proteomic Profiling and Analytical
Chemistry: The Crossroads (2nd Edition). Elsevier, 298 pages.

[2] David, L. N., & Michael, M. (2017). Cox, Principles of Biochemistry (7th
Edition). W. H. Freeman and Company, 3270 pages.

[3] Najafpour, G. (2015). Biochemical engineering and biotechnology (second

edition). Elsevier, 650 pages.

[4] Shu, G., Zhang, B., Zhang, Q., Wan, H., & Li, H. (2017). Effect of Temperature,
pH, Enzyme to Substrate Ratio, Substrate Concentration and Time on the Antioxidative
Activity of Hydrolysates from Goat Milk Casein by Alcalase. Acta Universitatis
Cibiniensis. Series E: Food Technology, 20(2), 29-38. https://doi.org/10.1515/aucft-
2016-0013

[5] Srinivasan, D., & Kirk, L. P. (2017). Fennema's Food Chemistry (5th Edition).
CRC press, 1107 pages.

[6] Thiansilakul, Y., Benjakul, S., & Shahidi, F. (2007). Compositions, functional
properties and antioxidative activity of protein hydrolysates prepared from round scad

14
(Decapterus maruadsi). Food Chemistry, 103(4), 1385-1394.
https://doi.org/10.1016/j.foodchem.2006.10.055

[7] Trần, B, L., Tôn, N. M, N, & Đinh, T. N.T. (2011). Thí nghiệm Hóa sinh Thực
phẩm. ĐHQG TP.HCM, 83 trang.

[8] Vo, T. D. L., & Pham, K. T. (2020). Copper-chelating peptide from salmon by-
product proteolysate. International Journal of Food Engineering.
https://doi.org/10.1515/ijfe-2019-0280

[9] Vo, T. D. L. (2018). Investigation of antioxidant activity of proteolysate derived

from Acetes japonicus. journal of science and Technology (JST-UD), 11(132), 15 141.

[10] Vo, T. D. L., Bui, A. N. N., Nguyen, T. V. V.. Nguyen, N. N. P., & Dang, H.
V. (2019). Investigation into antioxidant activity of protein hydrolysate derived from
white leg shrimp head (Litopenaeus vannamei). Journal of science and Technology (JST-
UD), 17(1.2), 75-79.

[11] Vo, T. D. L., Lam, H. H., Huynh, 0, N., & Nguyen, D, T. M. (2020).
Investigation of Calcium-Binding Capacity and Functional Properties of Acetes
Japonicus Protein Hydrolysate. Chemical engineering transactions, 78, 349-
354.https://doi.org/10.3303/CET2078059

[12] Vo. T. D. L., Pham, K. T., & Ha, D. Q. (2018). Recovery of Proteolysate from
Salmon By-Product: Investigation of Antioxidant Activity, Optimization of Hydrolysis,
Determination of Iron-Binding Activity And Identification of Bioactive Peptides. The
International Journal of Engineering and Science, 7(9), 18-30. 10.9790/1813-
0709041830

[13] Vo, T. D. L., Pham, K. T., Le, V. M. V, Lam, H. H., Huynh, 0. N., & Vo, B, C.
(2020). Evaluation of iron-binding capacity, amino acid composition, functional
properties of Acetes japonicus proteolysate and identification of iron-binding peptides.
Process Biochemistry,91, 374-386.
https://doi.org/10.1016/j.procbio.2020.01.007

15
 EXPERIMENT 5.2: COMPARISON OF INVERTASE HYDROLYSIS

AND ACIDIC HYDROLYSIS FOR SACCHAROSE

1. Comparison of catalysis capacity among enzyme and inorganic catalysts

1.1. Introduction

Inorganic catalysts efficiently function at high temperatures and high pressures, while
enzymes usually get denatured at higher temperatures (typically above 40°C). However,
there is an exception to this wherein enzymes that are isolated from organisms normally
living under extremely high temperatures are stable retaining their catalytic properties at
higher temperatures.

1.2. Principle

Most enzymes are proteins, which can increase the rate of chemical reactions.
Mechanism of enzyme catalysis is similar to that of other chemical catalysts in that the
crucial factor is a reduction of energy barrier(s) separating the reactants from the products.

As with other catalysts, the enzyme is not consumed or changed by the reaction (as a
substrate is) but is recycled such that a single enzyme performs many rounds of catalysis.

Enzymes function most efficiently within optimal temperature and pH range.

1.3. Apparatus

- Test tube, 10ml pipette

- Incubator, steamer/pot

1.4. Chemicals

- Invertase solution

- 5% saccharose solution

- Fehling reagent, 1% HCl solution

1.5. Procedure

Put 2ml of 5% saccharose solution into each of 4 numbered tubes, then add:

+ First tube: 1 ml of distilled water

+ Second tube: 1 ml of invertase solution

16
 + Third tube: 1ml of invertase solution

+ Fourth tube: 1ml of 1% HCI solution

Then place the first and second test tubes at room temperature, third tube at 55°C in
an incubator. Put the fourth tube in a boiling pot. After 10 minutes. take the test tubes out,
add 5ml of Fehling reagent per tube. Put all tubes in the pot for 2 minutes. Take out, cool
and compare the amount of precipitate of 𝐶𝑢 𝑂 formed in each tube.

1.6 Result and report

Amount of
Evaluation
Test precipitate
Substrate Catalyst Temperature, ℃ of Fehling
tube of 𝑪𝒖𝟐 𝑶
reaction
formed
1 Saccharose None 30
2 Saccharose Invertase 30
3 Saccharose Invertase 55
4 Saccharose HCl 100

2. Specificity of enzyme

2.1. Introduction
Specificity is a property of the enzyme and describes how restrictive the enzyme is
in its choice of substrate; a completely specific enzyme would have only one substrate.

The specificity of the serine proteases is usually not very high since they have
similar active sites and act through the same proteolytic mechanism.

Consequently, a single serine protease may act on various substrates although at

different rates. How the substrate fits the active site of the enzyme is of crucial
importance to the outcome of the enzyme-substrate reaction. The bond to be cleaved
must have a specific orientation relative to the amino acid side chains of the catalytic
triad. The most important factor governing the fit of a substrate for an enzyme is the
amino acid sequence around the bond to be cleaved.

Trypsin cleaves amides and esters of the basic amino acid arginine and lysine.
Thrombin has a similar preference, but is more specific for arginine than for lysine.

17
 2.2. Principle

Specificity is one of the most important properties of enzyme. A few enzymes exhibit
absolute specificity, that is, they will catalyze only one particular reaction one specific
substrate. Other enzymes will be specific for a particular type of chemical bond or
functional group. In general, there are four distinct types of specificity:

- Absolute specificity - one and only one substrate will fit with a particular

enzyme.

- Group specificity - the enzyme will act only on molecules that have specific

functional groups, such as amino, phosphate or alcohol groups.

- Linkage specificity - the enzyme will act on a particular type of chemical bond

regardless of the rest of the molecular structure.

- Stereochemical specificity - the enzyme will act on a particular steric or

optical isomer.

2.3. Apparatus

- Test tube, 10ml pipette

- Incubator, pot/steamer

2.4. Chemicals

- Amylase solution. invertase solution

- Lugol reagent, 1% gelatinized starch, 5% saccharose solution

- Fehling reagent

2.5. Procedure

Take 4 numbered tubes, put into first and second tube 5ml of 5% saccharose solution,
third and fourth tube 5ml of 1% starch adhesive. Then add 1 ml of invertase to each first
and third tube, 1 ml amylase in each second and fourth tube. Shake well and let them in
the incubator at 37℃ for 15 minutes. Take out, add 5ml of Fehling solution to each first
and second tube and place them into the boiling water bath for 2 minutes, take out, cool to
room temperature. Add 2 drops of Lugol reagents in each third and fourth tube.

18
 2.6. Result and report
Evaluation of Description (color,
Test tube Substrate Catalyst Fehling and Lugol turbidity,
tests coagulation…)
1 Saccharose Invertase
2 Saccharose Amylase
3 Starch Invertase
4 Starch Amylase

3. Questions

1. Present enzyme specificity.

Specificity is a property of the enzyme and describes how restrictive the enzyme is in
its choice of substrate; a completely specific enzyme would have only one substrate. A few
enzymes exhibit absolute specificity, that is, they will catalyze only one particular reaction
one specific substrate. Other enzymes will be specific for a particular type of chemical
bond or functional group. In general, there are four distinct types of specificity:

- Absolute specificity - one and only one substrate will fit with a particular

enzyme.

- Group specificity - the enzyme will act only on molecules that have specific

functional groups, such as amino, phosphate or alcohol groups.

- Linkage specificity - the enzyme will act on a particular type of chemical bond

regardless of the rest of the molecular structure.

- Stereochemical specificity - the enzyme will act on a particular steric or

optical isomer.

2. Give some examples of enzyme specificity.

 Example 1: Chymotrypsin and pepsin are able to act on almost any protein the
specificity of enzyme action, as they should if they are to act upon the differential
types of proteins consumed as food. Furthermore, since thrombin only interacts with
the protein fibrinogen, it is a part of a very delicate blood-clotting process that can
only react with one compound in order to keep the system working properly.

19
  Example 2: Lactase is an enzyme specific for the degradation of lactose into two
sugar monosaccharides, glucose and galactose. Another example is Glucokinase,
which is an enzyme involved in the phosphorylation of glucose to glucose-6-
phosphate. It is primarily active in the liver and is the main isozyme
of Hexokinase. Its absolute specificity refers to glucose being the only hexose that
is able to be its substrate, as opposed to hexokinase, which accommodates many
hexoses as its substrate.

3. Present kinetic parameters of enzymatic reaction.

Kinetic parameters of an enzymatic reaction can be determined by the Michaelis-

Menten equation:

𝑉 . [𝑆]
𝑉 =
𝐾 + [𝑆]

V0: the velocity of enzyme molecules that we can get

Vmax: the maximal velocity we can get when all the enzyme molecules are saturated
with substrate

[S]: Substrate concentration

Km: is the substrate concentration that provides half the maximal velocity.

20
 Figure 1: Michaelis-Menten curve

Source: tuitiontube.com

 At very low substrate concentration [S] compared to Km ([S] << Km), the graph
between Vo and [S] results as a straight line.

𝑉 . [𝑆]
𝑉 =
𝐾

 When Km = [S], then Vo = Vmax

𝑉 . [𝑆] 1
𝑉 = = 𝑉
2[𝑆] 2

 At very high substrate concentration [S] compared to Km ([S] >> Km), Vo approaches
to Vmax

𝑉 . [𝑆]
𝑉 = =𝑉
[𝑆]

21
 The Vmax value depends on environmental conditions, such as pH, temperature and
ionic strength. If the enzyme is multifunctional or if the reaction is reversible, we annotate
the Vmax for different reactions or for each direction of one reaction.

4. Present some enzyme mechanisms.

- Lock-and-key model:

Lock-and-key model is a model for enzyme-substrate interaction suggesting that

the enzyme and the substrate possess specific complementary geometric shapes that fit
exactly into one another. Enzymes are highly specific. They must bind to a specific
substrate before they can catalyze a chemical reaction.

A substrate is bond to the active site of the Enzyme by weak non-covalent bonds:

 hydrogen bonds

 hydrophobic interaction

 ionic interactions

 Van-der-Waals force

- Induced fit model:

The induced-fit model is a model for enzyme–substrate interaction to describe that
the substrate is capable of inducing the proper alignment of the active site of the enzyme,
causing the latter to subsequently perform its catalytic function.

A substrate is bond to the active site of the Enzyme by weak non-covalent bonds:
 hydrogen bonds
 hydrophobic interaction
 ionic interactions
 Van-der-Waals force

5. What is Fehling solution? How to prepare it?

Fehling’s solution, or Fehling’s reagent, is a chemical reagent that is used to

distinguish between an aldehyde and a ketone, other than α-hydroxy ketone. Practically,
it is used for the determination of reducing and non-reducing sugars that are present in

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carbohydrates. The test employed for this purpose is known as Fehling’s test. Fehling’s
solution cannot be used for aromatic aldehyde

In order to prepare Fehling solution, we combine two separate solutions: Fehling A

and Fehling B. Fehling A is a blue-colored aqueous solution of copper (II) sulfate (CuSO 4).
Fehling B is a colorless aqueous solution of potassium sodium tartrate (KNaC4H4O6·4H2O,
also known as Rochelle salt) in an alkaline base like sodium hydroxide (NaOH). The two
solutions are individually prepared and later mixed to give Fehling’s solution, which is
blue. In this final mixture, aqueous tartrate ions from the dissolved Rochelle salt bond to
Cu2+ (aq) ions from the dissolved copper sulfate crystals as bidentate ligands giving a
bistartratocuprate (II) complex.

4. References

[1] Ciborowski, P., & Silberring, J. (2016). Proteomic Profiling and Analytical
Chemistry: The Crossroads (2nd Edition). Elsevier, 298 pages.

[2] David, L. N., & Michael, M. (2017). Cox, Principles of biochemistry (7th
Edition). W. H. Freeman and Company, 3270 pages.

[3] Najafpour, G. (2015). Biochemical engineering and biotechnology (second

edition). Elsevier, 650 pages.

[4] Shu, G., Zhang, B., Zhang, Q., Wan, H., & Li, H. (2017). Effect of Temperature,
pH, Enzyme to Substrate Ratio, Substrate Concentration and Time on the Antioxidative
Activity of Hydrolysates from Goat Milk Casein by Alcalase. Acta Universitatis
Cibiniensis. Series E: Food Technology, 20(2), 29-38. https://doi.org/10.1515/aucft-
2016-0013

[5] Srinivasan, D., & Kirk, L. P. (2017). Fennema's Food Chemistry (5th Edition).
CRC press, 1107 pages.

[6] Thiansilakul, Y., Benjakul, S., & Shahidi, F. (2007). Compositions, functional
properties and antioxidative activity of protein hydrolysates prepared from round scad
(Decapterus maruadsi). Food Chemistry, 103(4), 1385-1394.
https://doi.org/10.1016/j.foodchem.2006.10.055

[7] Trần, B, L., Tôn, N. M, N, & Đinh, T. N.T. (2011). Thí nghiệm Hóa sinh Thực
phẩm. ĐHQG TP.HCM, 83 trang.

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 [8] Vo, T. D. L., & Pham, K. T. (2020). Copper-chelating peptide from salmon by-
product proteolysate. International Journal of Food Engineering.
https://doi.org/10.1515/ijfe-2019-0280

[9] Vo, T. D. L. (2018). Investigation of antioxidant activity of proteolysate derived

from Acetes japonicus. journal of science and Technology (JST-UD), 11(132), 15 141.

[10] Vo, T. D. L., Bui, A. N. N., Nguyen, T. V. V.. Nguyen, N. N. P., & Dang, H.
V. (2019). Investigation into antioxidant activity of protein hydrolysate derived from
white leg shrimp head (Litopenaeus vannamei). Journal of science and Technology (JST-
UD), 17(1.2), 75-79.

[11] Vo, T. D. L., Lam, H. H., Huynh, 0, N., & Nguyen, D, T. M. (2020).
Investigation of Calcium-Binding Capacity and Functional Properties of Acetes
Japonicus Protein Hydrolysate. Chemical engineering transactions, 78, 349-
354.https://doi.org/10.3303/CET2078059

[12] Vo. T. D. L., Pham, K. T., & Ha, D. Q. (2018). Recovery of Proteolysate From
Salmon By-Product: Investigation of Antioxidant Activity, Optimization of Hydrolysis,
Determination of Iron-Binding Activity And Identification of Bioactive Peptides. The
International Journal of Engineering and Science, 7(9), 18-30. 10.9790/1813-
0709041830

[13] Vo, T. D. L., Pham, K. T., Le, V. M. V, Lam, H. H., Huynh, 0. N., & Vo, B, C.
(2020). Evaluation of iron-binding capacity, amino acid composition, functional
properties of Acetes japonicus proteolysate and identification of iron-binding peptides.
Process Biochemistry, 91, 374-386. https://doi.org/10.1016/j.procbio.2020.01.007

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