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Introduction and Biomolecules

Subtopic:

Definition of biochemical engineering

Carbohydrates
Part 1 Amino acids and protein
The central Dogma of molecular biology
Buffer solution

1 2

What is Biochemical Engineering? What do Biochemical Engineer do?

Typical employers come

from all sectors of the
It is a subset of chemical engineering that mainly biotechnology industries,
deals with the design and construction of unit including those with
processes that involve biological organisms or interests in
molecules such as bioreactors. pharmaceuticals, food,
environment, waste
treatment, and
consulting.
3 4

Biochemical engineer responsibilities?

• To obtain the best • To separate the desired
biological catalyst products from the
(microorganism, animal reaction mixture in the
cell, plant cell, or most economical way
enzyme) for a desired
process.

• To create the best

possible environment for
the catalyst to perform by
designing the bioreactor
and operating it in the
most efficient way.
5 6

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What are carbohydrates?

 Major class of naturally occurring organic
compounds, including sugars, starches and
cellulose.

 Play key roles as structural and storage

compounds in cells. FUNCTIONS OF CARBOHYDRATE?

The formula for a carbohydrate is (CH2O)n, where

n≥ 3.

 Classified into THREE major groups :

monosaccharide, disaccharides and
polysaccharide. 7 8

Carbohydrates - The Functions

Most important source of energy for living

organisms.
 Linked to proteins or lipids.
 Form structural tissues in plants and in
microorganisms (cellulose, lignin, murein).
 Participates in biological transport, cell- cell
recognition, activation of growth factors, modulation
of the immune system.
 DNA and RNA framework.

9 10

Classifying carbohydrates Classifying carbohydrates

 Simple carbohydrates
1) Monosaccharides
Glucose, fructose, galactose
2) Disaccharides
Lactose, sucrose, maltose
 Complex carbohydrates
1) Polysaccharides
Starch, glycogen, cellulose

11 12
* chaining relise on 'bridging' of oxygen atoms - glycoside bonds

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Monosaccharides
 Smallest carbohydrates, also known as simple sugars. Aldose sugars
 Monosaccharides are categorized by:
1) number of carbons (typically 3-9)
2) whether an aldehyde or ketone

 Sugar containing an aldehydes is known as an aldose.

 Sugar containing a ketones is known as a ketose.

13 14

Aldose sugars Ketose sugars

15 16

Ketose sugars D and L notation

 D, L tells which of the two chiral isomers we are

referring to.

 If the –OH group on the next to the bottom

carbon atom points to the right , the isomer is a
D-isomer; if it points left, the isomer is L.

 The D form is usually the isomer found in

nature.

17 18

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D and L notation D and L notation

19 20

D notation Structural of representation of sugars

O
H C

H C OH Fisher projection: straight chain representation.

 Haworth projection: simple ring in perspective.

H C OH
 Conformational representation: chair and boat
C H 2 OH configurations.
Right = D

21 22

Rules of drawing Haworth projections

 Draw either a six or 5-membered ring including
oxygen as one atom.
O O

HAWORTH PROJECTION
 Most aldohexoses are six-membered.
 Aldotetroses, aldopentoses, ketohexoses are 5-
membered.

23 24

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Rules of drawing Haworth projections Rules of drawing Haworth projections

 Next: number the ring clockwise starting next to  For D-sugars the highest numbered carbon
the oxygen (furthest from the carbonyl) is drawn up. For L-
5 sugars, it is drawn down. - CH2OH
O O
4 1 4 1 For D-sugars, the OH group at the anomeric
position is drawn down for α and up for β. For L-
3 2 3 2 sugars α is up and β is down.

 If the substituent is to the right in the Fisher

projection, it will be drawn down in the Haworth
projection (Down-Right Rule)
25 26

Haworth Structure for D-Glucose Haworth Structure for D-Galactose

 Write –OH groups on the right (C2, C4) down.
Write –OH groups on the left (C3) up.
The new –OH on C1 has two possibilites: down
for  anomer, up for  anomer.

27 28

Isomers *homework Enantiomers

 Isomerism in which two isomers are mirror
 Isomers are molecules that have the same chemical formula but images of each other (D vs L).
different structures.
Stereoisomer differs in the 3-D orientation of atoms.
Diastereomers are isomers with > 1 chiral center.
– Pairs of isomers that have opposite configurations at one
or more of the chiral centers but that are not mirror
images of each other
Epimers are a special type of diastereomer.
– Stereoisomers with more than one chiral center which
differ in chirality at only one chiral center
– A chemical reaction which causes a change in chirality at
one of many chiral center is called an epimerisation

29 30

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Disaccharides

Disaccharides
Consist of 2 monosaccharides bonded
together.

31 32

Glycosidic Bond

 This is when two monosaccharides join to form a

Disaccharide.
 The reaction is similar to condensation.
 The reaction involves the water been given off.
Polysaccharides
>10 monosaccharides.

Most are made up of hundreds of

monosaccharides bonded together.

33 34

Cellulose

35 36

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Protein play key roles in a living system Amino acid: Basic unit of protein

 Almost all chemical reactions in a living cell are

catalyzed by protein enzymes biocalatalyst – faster
rate reaction
 Storage and transport of biochemical molecules,
such as oxygen, ions, and so on. Nutrients and
wastes are transported into and out of the cell -
transporter
 Physical cell support and shape (collagen).
 Regulatory and information transfer (hormones eg
insulin:).
 Mechanical movement (flagella, mitosis, muscles).
37 38

Examples of Amino acid Zwitterionic form of Amino Acids

 Zwitterion (dipolar ions) has both + and – charge

 Zwitterion is neutral overall
 Under normal cellular conditions amino acids are
zwitterions:
Amino group = -NH3+
Carboxyl group = -COO-

39 40

pH and Ionization The peptide bond ( CO - NH linkage)

Dipeptide
Tripeptide
Peptides (4-50 amino acids)
Proteins (more than 50 amino
41 acids) 42

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Primary Structure of Proteins

 Primary structure of a proteins is the particular
sequence of amino acids connected by peptide bonds.

Proteins are linear polymers of amino acids

43 44

Secondary Structure Basic structural units of proteins:

Secondary structure
 Secondary structure of a protein is the arrangement
of polypeptide backbone of the protein in space.
 The secondary structure includes two kinds of
repeating pattern known as helixes (α-helix, triple
helix) and sheet (β-sheet).
 Hydrogen bonding between backbone atoms are
responsible for both of these secondary structures.

Helix (α-helix, triple helix)

Sheet (β-sheet).
45 46

Tertiary Structure Tertiary Structure

 The overall three dimensional shape that results
from the folding of a protein chain.
 Tertiary structure depends mainly on interactions
of amino acid R groups that are far apart along the
same backbone.
 Cross links between R groups of amino acids in
chain:
disulfide –S–S– +
ionic –COO– H3N–
H bonds C=O HO–
hydrophobic –CH3 H3C–

47 48

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Quaternary Structure
• The way in which two or more polypeptide
chains associate to form a single three-
dimensional protein unit. Non-covalent
forces are responsible for quaternary
structure essential to the function of
proteins.
• Example is hemoglobin that carries oxygen
in blood.
-Four polypeptide chains
49 50

Protein Hydrolysis Hydrolysis of Dipeptide

• Break down of peptide bonds OH

• Requires acid or base, water and heat CH 3 O CH 2 O H 2O, H
+
+
• Gives smaller peptides and amino acids H 3 N CH C N CH C OH
• Similar to digestion of proteins using H heat
enzymes OH
• Occurs in cells to provide amino acids to CH2 O
CH 3 O +
synthesize other proteins and tissues +
H 3 N CH COH + H3 N CH C OH

51 52

Denaturation Application of Denaturation

Disruption of secondary, tertiary and quaternary • Hard boiling an egg

protein structure by • Wiping the skin with alcohol swab for
heat/organics
injection
Break apart H bonds and disrupt hydrophobic
attractions • Cooking food to destroy E. coli.
acids/ bases
• Heat used to cauterize blood vessels
Break H bonds between polar R groups and
ionic bonds • Autoclave sterilizes instruments
heavy metal ions • Milk is heated to make yogurt
React with S-S bonds to form solids
agitation
Stretches chains until bonds break
53 54

9
 What is DNA and RNA?
CHAPTER 1
 DNA – Deoxyribonucleic acid
 RNA – Ribonucleic acid
INTRODUCTION & BIOMOLECULES  DNA stores and preserves genetic
information
Part 2  RNA plays a central role in protein synthesis
 Both DNA and RNA are large polymers made
By
of their corresponding nucleotides
LAW JENG YIH (condensation)

Introduction
The Central Dogma Nucleotide
of Molecular Biology
 Building block of DNA and RNA
Cell  Consists of 3 major components:
1) phosphoric acid
Transcription DNA 2) pentose (5-carbon sugar)
mRNA -ribose or deoxyribose
Translation Ribosome 3) base (purine or pyrimidine)

Polypeptide
(protein)
©1998 Timothy G. Standish

A Nucleotide
Adenosine Mono Phosphate (AMP)
CENTRAL DOGMA?
OH
 Explains how the information stored in the Phosphate
- NH2
cell is translated to the synthesis of various HO P O Base
proteins, and these proteins control various N
cellular activities. H+ H
O N
H
N N
5’CH2
 Briefly, the information in DNA is transcribed O
4’ 1’
to RNA and this information is translated to a Sugar Nucleoside
linear sequence of proteins. 3’ 2’

3
OH H
OH

1
 Purines Pyrimidines Base Pairing
Adenine And Cytosine
NH2 O O
Thymine Uracil
Adenine CH3 (DNA) (RNA)
N NH NH
N +
N N N O N O
O -
NH2
Guanine
N Cytosine -
NH
N
N N NH2 N O

Base Pairing Base Pairing

Guanine And Cytosine Guanine And Thymine

-
+ -

+ - +

+ -
+

The Central Dogma

Base Pairing
Adenine And Thymine

DNA Proposed by Francis Crick in 1958 to describe the

flow of information in a cell.
+ - Information stored in DNA is transferred
Adenine Thymine residue-by-residue to RNA which in turn transfers
the information residue-by-residue to protein.
RNA
- + The Central Dogma was proposed by Crick to
help scientists think about molecular biology. It
has undergone numerous revisions in the past 45
years.
Protein

2
The Central Dogma
DNA: terminology

DNA deoxyribonucleic acid

RNA

Protein

DNA DNA: structure

base: thymine
monophosphate (pyrimidine)

1. DNA is double stranded

 sugar: 2’-deoxyribose 2. DNA strands are antiparallel
3. G-C pairs have 3 hydrogen bonds
4. A-T pairs have 2 hydrogen bonds
5’ 5. One strand is the complement of the other
6. Major and minor grooves present different
4’ 1’ surfaces
(5’ to 3’) 3’ 2’ 7. Cellular DNA is almost exclusively B-DNA
3’ linkage 8. B-DNA has ~10.5 bp/turn of the helix

base:adenine
(purine)
5’ linkage

no 2’-hydroxyl

The Central Dogma

DNA: terminology
base

DNA
sugar

nucleoside

RNA ribonucleic acid

base
phosphate(s)

sugar

Protein

nucleotides (nucleoside mono-, di-, and triphosphates)

3
 RNA: terminology How does RNA differ from DNA?

 There are three important differences

between RNA and DNA:
Base Nucleoside (RNA) Deoxynucleoside (DNA)
base
Adenine Adenosine Deoxyadenosine
 (1) the sugar in RNA is ribose instead
Guanine Guanosine Deoxyguanosine
of deoxyribose,
sugar
Cytosine Cytidine Deoxycytidine  (2) RNA is generally single-stranded
Uracil Uridine (not usually found)
and not double-stranded, and
nucleoside Thymine (not usually found) (Deoxy)thymidinea
 (3) RNA contains uracil in place of
3 major types of RNA thymine.
1) messenger RNA (mRNA); template for protein synthesis
2) transfer RNA (tRNA); adaptor molecules that decode the genetic code
3) ribosomal RNA (rRNA); catalyzing the synthesis of proteins 22

RNA: Structure

1. RNA can be single or double stranded

2. G-C pairs have 3 hydrogen bonds
3. A-U pairs have 2 hydrogen bonds
4. Single-stranded, double-stranded, and loop RNA
present different surfaces 23

RNA: Structure The Central Dogma

Primary structure

DNA

Secondary structure Tertiary structure

C

RNA
D

E G

F
B
A
Protein

4
 20 amino acids
carboxyl group

amino group

 DNA carries genetic information in its base

sequence
 The genetic information in DNA is transcribed
by RNA molecules and translated in protein
synthesis

Protein structure The central of Dogma

 We first examine the simplest way of

looking at protein synthesis as expressed in
the so called Central Dogma of Biology,
namely that the direction of information
flow in the cell is from DNA to mRNA to
proteins.

-helix antiparallel -sheet 29

The Central Dogma ATGAGTAACGCG

(gene)
TACTCATTGCGC RNA Synthesis
Replication
duplication of DNA using DNA as the template
ATGAGTAACGCG
DNA TACTCATTGCGC  How does the cell make RNA?
+
(nontemplate, antisense) ATGAGTAACGCG
(template, sense) TACTCATTGCGC
Transcription
synthesis of RNA using DNA as the template
 In transcription, segments of DNA serve as
templates to produce complementary RNA
RNA (mRNA) AUGAGUAACGCG
codon molecules.
tRNA
ribosomes
Translation
(protein) MetSerAsnAla
synthesis of proteins using RNA as the template
Protein

30

5
Transcription
 Most of the work of making RNA takes
place during transcription. During
transcription, segments of DNA serve as
templates to produce complementary
RNA molecules.
 The base sequences of the transcribed
RNA complement the base sequences of
the template DNA.
31
The role of RNA (Translation)
 Genes contain coded DNA instructions that
tell cells how to build proteins.
 The first step in decoding these genetic
instructions is to copy part of the base
sequence from DNA into RNA.
 RNA, like DNA, is a nucleic acid that consists
of a long chain of nucleotides.
 RNA then uses the base sequence copied
from DNA to direct the production of
proteins. 32
The role of RNA (Translation) - cont
 The roles played by DNA and RNA are
similar to the master plans and blueprints
used by builders.
33
The role of RNA (Translation) - cont
 A master plan has all the information
needed to construct a building. Builders
never bring a valuable master plan to the
building site, where it might be damaged
or lost. Instead, they prepare inexpensive,
disposable copies of the master plan
called blueprints.
34
The role of RNA (Translation) - cont
 Similarly, the cell uses DNA “master plan”
to prepare RNA “blueprints.”
 The DNA molecule stays safely in the cell’s
nucleus, while RNA molecules go to the
protein-building sites in the cytoplasm—
the ribosomes.
35
The role of RNA (Translation) - cont
 You can think of an RNA molecule, as a
disposable copy of a segment of DNA, a
working copy of a single gene.
 RNA has many functions, but most RNA
molecules are involved in protein
synthesis only.
 RNA controls the assembly of amino acids
into proteins. Each type of RNA molecule
36
specializes in a different aspect of this job.
6
Function of RNA Ribosomal of RNA

 Proteins are assembled

– The three main types of RNA are on ribosomes, small
messenger RNA, ribosomal RNA, and organelles composed of
transfer RNA. two subunits.

 These ribosome subunits

are made up of several
ribosomal RNA (rRNA)
molecules and as many as
37
80 different proteins. 40

Messenger of RNA Transfer of RNA

 Most genes contain
instructions for
assembling amino acids  You will see that the tRNA molecules
into proteins. have a set of three nucleotide bases at
one end that are complementary to a
 The RNA molecules that corresponding codon. The bases on
carry copies of these the tRNA are called the anti codon.
instructions are known
as messenger RNA  This is critical because the anti
(mRNA): They carry codons make the connection between
information from DNA the codons and the correct amino
to other parts of the cell. acids that go with each codon.
38 41

Messenger of RNA Transfer of RNA

 A critical feature of mRNA and how it is  When a protein is

translated is the fact that each three built, a transfer RNA
nucleotides in the mRNA is called a codon and (tRNA) molecule
it is the codon that is translated. transfers each amino
 Thus the sequence of codons corresponds to acid to the ribosome
the sequence of amino acids in the polypeptide. as it is specified by the
coded messages in
mRNA.

39 42

7
Buffer Solutions
 A buffer is a solution characterised by the
ability to resist changes in pH when limited
amounts of acid or base are added to it.
 Buffers contain either a weak acid and its
conjugate base or a weak base and its
conjugate acid.
 Thus, a buffer solution contains both an acid
species and a base species in equilibrium.
Buffer Solutions
 In a solution there is an equilibrium between a weak
acid, HA, and its conjugate base, A-.
HA + H2O H3O+ + A−
 When hydrogen ions are added to the solution the
equilibrium moves to the left, in accordance with Le
Chatelier's principle, as there are hydrogen ions on the
right-hand side of the equilibrium expression. When
hydroxide ions are added the equilibrium moves to the
right as hydrogen ions are removed in the reaction H+ +
OH- → H2O.
Buffer Solutions
 The acid dissociation constant for a weak
acid, HA, is defined as
 Simple manipulation with logarithms gives the
Henderson-Hasselbalch equation, which
describes pH in terms of pKa
Buffer Solutions
 In this equation [A−] is the concentration of the
conjugate base and [HA] is the concentration of the
acid. It follows that when the concentrations of acid
and conjugate base are equal, often described as
half-neutralization, pH = pKa.
 Maximum buffering capacity is found when pH = pKa,
and buffer range is considered to be at pH = pKa±1.
 In general a buffer solution may be made up of more
than one weak acid and its conjugate base; if the
individual buffer regions overlap a wider buffer region
is created by mixing the two buffering agents.
Buffer Solutions
Consider an equal molar mixture of CH3COOH and CH3COONa
CH3COOH (aq) H+ (aq) + CH3COO- (aq)
Adding more acid creates a shift left IF
enough acetate ions are present
16.3
HCl H+ + Cl-
HCl + CH3COO- CH3COOH + Cl-
16.3
8
Applications Summary
 Their resistance to changes in pH makes buffer solutions  The receptors on the surface of the cell are
very useful for chemical manufacturing and essential for
primarily carbohydrates. They are highly
many biochemical processes. The ideal buffer for a
particular pH has a pKa equal to that pH, since such a specific and receive molecules destined to
solution has maximum buffer capacity. enter the cell.
 Buffer solutions are necessary to keep the correct pH for  The information structure of the cell is found
enzymes in many organisms to work. Many enzymes
work only under very precise conditions; if the pH strays
in nucleotides.
too far out of the margin, the enzymes slow or stop
working and can denature, thus permanently disabling its
catalytic activity.

Applications
 A buffer of carbonic acid (H2CO3) and bicarbonate
(HCO3−) is present in blood plasma, to maintain a pH
between 7.35 and 7.45.
 Industrially, buffer solutions are used in fermentation
processes and in setting the correct conditions for dyes
used in colouring fabrics. They are also used in chemical
analysis and calibration of pH meters.
 Majority of biological samples that are used in research
are made in buffers specially PBS (phosphate buffer
saline) at pH 7.4.

Summary
 Biological systems are composed of carbon,
oxygen, hydrogen, and notrogen.(Note: some
cellular molecules contain metals and sulfur is
frequently present in disulfide bonds.)
 These molecules are used to build lipids, proteins,
carbohydrates, and nucleic acids, the building
blocks for cell structure and chemical reagents for
cell function.
 Proteins conduct the business of the cell by
regulating cell function.
 Carbohydrates serve primarily as energy sources.

9
Biochemical Engineering
CPB 30103
By
LAW JENG YIH
Outlines
1) Nature of enzyme action
2) Michaelis-Menten Equation,
Lineweaver-Burk, & other plots
3) Steady state approach
4) Factors affecting reaction rates
5) Enzyme inhibition; competitive &
non-competitive inhibition.
Enzymes
 The interaction between enzyme and
substrate is very specific. Specificity may be
for a class of compounds or for a particular
compound. Eg. Hexokinase / glucokinase
 Active site - A pocket in an enzyme with the
specific shape and chemical makeup
necessary to bind a substrate and where the
reaction takes place
 Substrate - A reactant in an enzyme
catalyzed reaction
 Activity lost if denatured
CHAPTER 2
ENZYME KINETICS AND
INDUSTRIAL ENZYMOLOGY
Enzymes
 Enzymes are proteins that act as catalyst for
a biochemical reaction (biological catalysts)
 They are not consumed or altered during
the reaction
 They do not change the equilibrium, just
reduce the time required to reach
equilibrium.
 They increase the rate of a reaction by
lowering the activation energy barrier.
 The reactant usually binds to the enzyme
forming a transition complex that stabilizes
the transition state.
Energy of Activation
 In a chemical reaction, the reactants must
absorb a certain amount of energy from the
environment before a reaction can take
place.
 The specific amount of energy required for
the reaction to proceed is called the ENERGY
OF ACTIVATION.
1
 Energy of Activation Energy of Activation

 In reactions occurring outside of living  To overcome the activation energy required

organisms, simply adding heat can
provide activation energy.
 Inside of living organisms, however,
another method must be used. Heat is
 With a lower energy requirement, more
very harmful to the cells and proteins
of plants and animals.
for certain reactions to take place, living
organisms employ enzymes. Enzymes
function by being able to LOWER the
activation energy needed in specific
reactions.
molecules will be able to react with each
other and the reaction can swiftly occur at
temperatures able to support life.

Energy of Activation
Collision of certain
molecules across
certain potential energy
barrier.
Transition state propose
unstable intermediate
complex where the
bonds of S are distorted
sufficiently, so
conversion to P is
possible.
S: Substrate
P: Product
Enzyme Reactions vs
Chemical Reactions
 Highly specific - catalyze only one or small
number of chemical reactions
 Their rate of reaction is usually much faster
than non-biological catalyst
 The reaction conditions ( T, P, pH) are very
mild.
 Enzymes are comparatively sensitive or
unstable molecules and require care in their
use.

Name of Enzymes
Name of Enzymes *Non-descriptive name such as:

rennin curding of milk to start cheese-making processer

 End in –ase pepsin hydrolyzes proteins at acidic pH
 Identifies a reacting substance trypsin hydrolyzes proteins at mild alkaline pH
sucrase – reacts sucrose *Name of substrate + ase :
lipase - reacts lipid Substrate Enzyme Product
 Describes function of enzyme glucose + maltose +
Starch α-amylase
oxidase – catalyzes oxidation oligosaccharides
lactose lactase glucose + galactose
hydrolase – catalyzes hydrolysis
fat lipase fatty acids + glycerol
 Common names of digestion enzymes still use – maltose maltase glucose
in pepsin, trypsin
urea + H2O urease 2NH3 + CO2
cellobiose cellobiase glucose

2
 Cofactor or Coenzymes
 Coenzymes are organic molecules that are required by
certain enzymes to carry out catalysis.
 They bind to the active site of the enzyme
and participate in catalysis but are not considered
substrates of the reaction.
 coenzymes often function as intermediate carriers of
electrons, specific atoms or functional groups that are
transferred in the overall reaction.
 An example of this would be the role of NAD in the
transfer of electrons in certain coupled oxidation
reduction reactions.
Cofactor or Coenzymes
 Cofactors are often classified as inorganic substances
that are required for increasing the rate of catalysis.
 Two ways of Cofactor function:
 Activates the enzymes by changing its 3-D shape to
provide a shape that maximizes the binding and
interaction of the enzymes with the substrate
 Participates in the overall reaction as another substrate
(coenzymes mainly). In this case, it commonly acts as a
donor or acceptor of a particular chemical group (group
transfer agent)

Examples of Classification of
Classification of Enzymes Enzymes

Class Reactions catalyzed  Oxidoreductoases

 Oxidoreductases oxidation-reduction oxidases - oxidize ,reductases – reduce
 Transferases transfer group of atoms  Transferases
 Hydrolases hydrolysis transaminases – transfer amino groups
 Lyases add/remove atoms to/from a kinases – transfer phosphate groups
double bond  Hydrolases
 Isomerases carry out many kinds of proteases - hydrolyze peptide bonds
isomerization, eg. L to D lipases – hydrolyze lipid ester bonds
isomerizations
 Lyases
 Ligases Catalyze reactions in which two
chemical groups are joined with carboxylases – add CO2
the use of energy from ATP hydrolases – add H2O

Enzyme Action:
Lock and Key Model Lock and Key Model
 An enzyme binds a substrate in a region called
the active site.
 Only certain substrates can fit the active site.
 Amino acid R groups in the active site help P
substrate bind. + S +
S
 Enzyme-substrate complex forms.
P
 Substrate reacts to form product.
 Product is released.
E + S ES complex E + P

3
 Enzyme Action:
Induced Fit Model
 Enzyme structure flexible, not rigid.
 Enzyme and flexible active site adjust shape to bind
substrate.
 This sudden change in shape can lead to the
breaking of bonds within a single substrate
molecule, forming two new molecules. Conversely,
it can also bring two-substrate molecules close
enough together for them to bond with each other,
forming one new molecule.
 Increases range of substrate specificity.
 Shape changes also improve catalysis during
reaction.

Enzyme Action: Enzyme Action:

Induced Fit Model Induced Fit Model

P
S
S
P

E + S ES complex E + P

Formula for a simple enzyme-

Enzyme Kinetics catalyzed reaction

 Study of the rates of enzyme-catalyzed

reactions
 Provides information on enzyme
specificities and mechanisms

4
Formula for a simple enzyme-
catalyzed reaction Enzyme-substrate complex
 Leonor Michaelis (1913): noticed that at constant
[enzyme] the rate of a reaction increases with
 E = free enzyme increasing [S] until a “maximal velocity” (Vmax) is
achieved
 S = substrate
 This “saturation effect” is an important distinction
 ES = enzyme-substrate complex versus uncatalyzed reactions
 P = product  Interpretation of data: ES complexes formed until
substrate saturation occurs at which point no more
substrate binding sites (i.e., enzyme molecules) are
available

Significance of MM Equation
 Km has a unit of concentration. It is a constant for every
enzyme under specific conditions
 When [S] <<< Km, Vo is directly proportional to [S]
 When [S] >>> Km, Vo = Vmax
 When [S] = Km, Vo = ½ Vmax
 The Km value of an enzyme indicates the concentration of the
substrate required for significant catalysis
 Since Km = (k2+k-1)/k1, when k-1 >>> k2, Km = k-1/k1
 Dissociation constant of ES, KES = [E][S] / [ES] = k-1/k1
 When k-1 >>> k2, Km = KES. High Km = high dissociation = weak
binding between E and S
 When [E]T = [ES], Vmax = k2[E]T.
k2 is called the turnover number: the rate when enzyme is
saturated with substrate

Line-Weaver Burk Plot

 Also called the double-reciprocal plot

 1/Vo = (Km/Vmax).(1/[S]) + 1/Vmax
 This is equation for a straight line y = mx + c  Kindly refer Chapter 2 Part 2 for further
 Slope = Km/Vmax details relating to the equation
 Y-intercept = 1/Vmax derivation on Michaelis-Menten
 X-intercept = -1/Km Equation, Lineweaver-Burk, & other
 Useful for experimental determination of Km plots as well as steady state approach
and Vmax

5
 Factors Affecting Reaction
Enzyme Activity Rates

 Enzymes are required in minute quantities and
enhance reaction rates by 1010- to 1020-fold
 When the enzyme is part of a crude preparation, its
concentration is in terms of units
 Enzyme activity is the ability of an enzyme to
modify a reactant. 1 unit (U) is the enzyme activity
that converts 1 μmole of reactant per min under
standard conditions.
 The specific activity of an enzyme is defined as
the activity per unit of mass or U/mg protein
 The study of enzyme reaction rates is called
enzyme kinetics. Enzyme kinetics are
affected by:
 Temperature and pH: Each enzymatic
reaction has an optimum pH and optimum
temperature. Extreme temperature or pH
disrupts enzyme structure and therefore
reaction rate
 Substrate concentration: The reaction rate =
k [P] / [S]. The rate can be increased by
adding more substrate, or by removing
product as it is formed

Factors Affecting Enzyme Factors Affecting Enzyme

Action: Temperature Action

 Little activity at low temperature Optimum temperature

 Rate increases with temperature
 Most active at optimum temperatures
Reaction
(usually 37°C in humans) Rate
 Activity lost with denaturation at high
temperatures

Low High
Temperature

Factors Affecting Enzyme Action: Factors Affecting Enzyme

Substrate Concentration Action

 Increasing substrate concentration

Maximum activity
increases the rate of reaction (enzyme
concentration is constant)
 Maximum activity reached when all of Reaction
enzyme combines with substrate Rate

substrate concentration

6
 Factors Affecting Enzyme Factors Affecting Enzyme
Action: pH Action

 Maximum activity at optimum pH

 R groups of amino acids have proper
charge
 Narrow range of activity Reaction
Rate
 Most lose activity in low or high pH
Optimum pH

3 5 7 9 11

pH

Enzyme Inhibition Enzyme Inhibition

Inhibitors
 Cause a loss of catalytic activity
 Change the protein structure of an enzyme
 May be a reversible or irreversible inhibition
 Specific enzyme inhibitors regulate enzyme
activity and help us understand mechanism of
enzyme action. (Denaturing agents are not
inhibitors)
Inhibitors
 Irreversible inhibitors form covalent or very
tight permanent bonds with the active site of the
enzyme and incapacitating the enzyme. 3
classes: group-specific reagents, substrate
analogs, suicide inhibitors
 Reversible inhibitors form an EI complex that
can be dissociated back to enzyme and free
inhibitor. 3 groups based on their mechanism of
action:
competitive, non-competitive and uncompetitive.

Competitive Inhibition Competitive Inhibition

A competitive inhibitor
 Has a structure similar to substrate

 Occupies active site

 Competes with substrate for binding to enzyme

(active site)
 E + S = ES or E + I = EI . Both S and I cannot bind
enzyme at the same time
 In presence of I, the equilibrium of E + S = ES is
shifted to the left causing dissociation of ES.
 This can be reversed / corrected by increasing [S]

 Vmax is not changed, Km is increased

7
 Noncompetitive Inhibition
A noncompetitive inhibitor
 Inhibitor binding site is distinct from substrate binding
site, it does not have a structure like substrate
 Binds to the enzyme but not active site
 Changes the shape of enzyme and active site
 Substrate cannot fit altered active site
 Can bind to free enzyme E and to ES
 E + I = EI, ES + I = ESI or EI + S = ESI
 Both EI and ESI are enzymatically inactive
 The effective functional [E] (and [S]) is reduced
 Reaction of unaffected ES proceeds normally
 Inhibition cannot be reversed by increasing [S]
 Km is not changed, Vmax is decreased

8
 CPB 30103: Biochemical Engineering
By Law Jeng Yih

ENZYME KINETICS AND INDUSTRIAL ENZYMOLOGY

Enzyme Structure
Enzymes are also classified on the basis of their composition. Enzymes composed wholly
of protein are known as simple enzymes in contrast to complex enzymes, which are
composed of protein plus a relatively small organic molecule. Complex enzymes are also
known as holoenzymes. In this terminology the protein component is known as the
apoenzyme, while the non-protein component is known as the coenzyme or prosthetic
group where prosthetic group describes a complex in which the small organic molecule
is bound to the apoenzyme by covalent bonds; when the binding between the apoenzyme
and non-protein components is non-covalent, the small organic molecule is called a
coenzyme. Many prosthetic groups and coenzymes are water-soluble derivatives of
vitamins. It should be noted that the main clinical symptoms of dietary vitamin
insufficiency generally arise from the malfunction of enzymes, which lack sufficient
cofactors derived from vitamins to maintain homeostasis.

The non-protein component of an enzyme may be as simple as a metal ion or as

complex as a small non-protein organic molecule. Enzymes that require a metal in their
composition are known as metalloenzymes if they bind and retain their metal atom(s)
under all conditions with very high affinity. Those which have a lower affinity for metal
ion, but still require the metal ion for activity, are known as metal-activated enzymes.

Enzyme-Substrate Interactions
The favored model of enzyme substrate interaction is known as the induced fit model.
This model proposes that the initial interaction between enzyme and substrate is
relatively weak, but that these weak interactions rapidly induce conformational changes
in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds to
be altered. After binding takes place, one or more mechanisms of catalysis generates
transition- state complexes and reaction products. The possible mechanisms of catalysis
are four in number:

1. Catalysis by Bond Strain: In this form of catalysis, the induced structural

rearrangements that take place with the binding of substrate and enzyme ultimately
produce strained substrate bonds, which more easily attain the transition state. The new
conformation often forces substrate atoms and bulky catalytic groups, such as aspartate
and glutamate, into conformations that strain existing substrate bonds.

2. Catalysis by Proximity and Orientation: Enzyme-substrate interactions orient

reactive groups and bring them into proximity with one another. In addition to inducing
strain, groups such as aspartate are frequently chemically reactive as well, and their
proximity and orientation toward the substrate thus favors their participation in catalysis.

1
 CPB 30103: Biochemical Engineering
By Law Jeng Yih

3. Catalysis Involving Proton Donors (Acids) and Acceptors (Bases): Other

mechanisms also contribute significantly to the completion of catalytic events initiated by
a strain mechanism, for example, the use of glutamate as a general acid catalyst (proton
donor).

4. Covalent Catalysis: In catalysis that takes place by covalent mechanisms, the

substrate is oriented to active sites on the enzymes in such a way that a covalent
intermediate forms between the enzyme or coenzyme and the substrate. One of the best-
known examples of this mechanism is that involving proteolysis by serine proteases,
which include both digestive enzymes (trypsin, chymotrypsin, and elastase) and several
enzymes of the blood clotting cascade. These proteases contain an active site serine
whose R group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond,
thereby causing hydrolysis of the peptide bond.

Chemical Reactions and Rates

According to the conventions of biochemistry, the rate of a chemical reaction is described
by the number of molecules of reactant(s) that are converted into product(s) in a specified
time period. Reaction rate is always dependent on the concentration of the chemicals
involved in the process and on rate constants that are characteristic of the reaction. For
example, the reaction in which A is converted to B is written as follows:

A ------> B

The rate of this reaction is expressed algebraically as either a decrease in the

concentration of reactant A:

-[A] = k[B]

or an increase in the concentration of product B:

[B] = k[A]

In the second equation (of the 3 above) the negative sign signifies a decrease in
concentration of A as the reaction progresses, brackets define concentration in molarity
and the k is known as a rate constant. Rate constants are simply proportionality constants
that provide a quantitative connection between chemical concentrations and reaction
rates. Each chemical reaction has characteristic values for its rate constants; these in turn
directly relate to the equilibrium constant for that reaction. Thus, reaction can be
rewritten as an equilibrium expression in order to show the relationship between reaction
rates, rate constants and the equilibrium constant for this simple case. The rate constant
for the forward reaction is defined as k+1 and the reverse as k-1.

2
 CPB 30103: Biochemical Engineering
By Law Jeng Yih

At equilibrium the rate (V) of the forward reaction (A ----> B) is, by definition,
equal to that of the reverse or back reaction (B ----> A), a relationship which is
algebraically symbolized as:

Vforward = Vreverse
where, for the forward reaction:

Vforward = k+1[A]
and for the reverse reaction:

Vreverse = k-1[B]

In the above equations, k+1 and k-1 represent rate constants for the forward and
reverse reactions, respectively. The negative subscript refers only to a reverse reaction,
not to an actual negative value for the constant. To put the relationships of the two
equations into words, we state that the rate of the forward reaction [Vforward] is equal to
the product of the forward rate constant k+1 and the molar concentration of A. The rate of
the reverse reaction is equal to the product of the reverse rate constant k-1 and the molar
concentration of B.

At equilibrium, the rate of the forward reaction is equal to the rate of the reverse
reaction leading to the equilibrium constant of the reaction and is expressed by:

Keq = k+1/ k-1 = [B]/[A]

This equation demonstrates that the equilibrium constant for a chemical reaction
is not only equal to the equilibrium ratio of product and reactant concentrations, but is
also equal to the ratio of the characteristic rate constants of the reaction.

Inhibition of Enzyme Catalyzed Reactions

Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menton
equation are useful in the analysis of enzyme inhibition.

As noted earlier, high concentrations of substrate can displace virtually all

competitive inhibitor bound to active sites. Thus, it is apparent that Vmax should be
unchanged by competitive inhibitors. This characteristic of competitive inhibitors is
reflected in the identical vertical-axis intercepts of Lineweaver-Burk plots, with and
without inhibitor.

3
 CPB 30103: Biochemical Engineering
By Law Jeng Yih

Lineweaver-Burk Plots of Inhibited Enzymes

Since attaining Vmax requires appreciably higher substrate concentrations in the

presence of competitive inhibitor, Km (the substrate concentration at half maximal
velocity) is also higher, as demonstrated by the differing negative intercepts on the
horizontal axis in panel B.

Analogously, panel C illustrates that noncompetitive inhibitors appear to have no

effect on the intercept at the x-axis implying that noncompetitive inhibitors have no effect
on the Km of the enzymes they inhibit. Since noncompetitive inhibitors do not interfere in
the equilibration of enzyme, substrate and ES complexes, the Km's of Michaelis-Menten
type enzymes are not expected to be affected by noncompetitive inhibitors, as
demonstrated by x-axis intercepts in panel C. However, because complexes that contain
inhibitor (ESI) are incapable of progressing to reaction products, the effect of a
noncompetitive inhibitor is to reduce the concentration of ES complexes that can advance
to product. Since Vmax = k2[Etotal], and the concentration of competent Etotal is diminished
by the amount of ESI formed, noncompetitive inhibitors are expected to decrease Vmax, as
illustrated by the y-axis intercepts in panel C.

4
 CPB 30103: Biochemical Engineering
By Law Jeng Yih

A corresponding analysis of uncompetitive inhibition leads to the expectation that

these inhibitors should change the apparent values of Km as well as Vmax. Changing both
constants leads to double reciprocal plots, in which intercepts on the x and y axes are
proportionately changed; this leads to the production of parallel lines in inhibited and
uninhibited reactions.

Linearizing the Michaelis-Menten Equation

Three ways in which the hyperbolic relationship between the initial rate of reaction and
the initial substrate concentration

can be rearranged to give linear plots. The examples are drawn using Km = 2 mM and
Vmax = 4 µM min-1.

(a) Lineweaver-Burk (double-reciprocal) plot of 1/v against 1/[S]0 giving intercepts at

1/Vmax and -1/Km

5
 CPB 30103: Biochemical Engineering
By Law Jeng Yih

(b) Eadie-Hofstee plot of v against v/[S]0 giving intercepts at Vmax and Vmax/Km

c) Hanes-Woolf (half-reciprocal) plot of [S]0/v against [S]0 giving intercepts at Km/Vmax

and Km.

6
 Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

Enzymology consists on the study of enzyme mechanisms

It typically involves the following steps:

1) Determination of the rate of catalysis as a function of several

experimental variables (enzyme kinetics)

2) Determination of the 3-D structure of the Enzyme, Enzyme-Substrate

Complex, and of the Enzyme-Transition-State-Analog Complex

3) Isotopic Effects

4) Site-Directed Mutagenesis of the Catalytic Residues

Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

The rate of catalysis of an enzyme depends on substrate concentration The dependence of V0 on the
(the higher [S] the higher the rate) initial concentration of S is
complex
In an enzymatic reaction the [S] changes with time as the enzyme goes through
many turnovers and substrate is converted onto product 1) It approximates a rectangular
-> Difficult to study the [S] dependence hyperbola
2) At relatively low [S] the
A simple approach is to study the rate of the reaction at early times (initial V0 increases almost linearly with
conditions) when [S]>>>[E], so that [S] can be assumed to be constant and equal to increases in [S] (pseudo-first-
the initial concentration used in the assay ([S]0). The measured velocity at early order)
times is termed V0. 3) At high [S] V0 approximates
asymptotically to a constant value
Enzyme kinetics experiments typically consist of measuring the velocity of the termed Vmax
reaction at early times for several initial concentrations of substrate, which all 4) The curve is similar to a simple
of them are much higher than the concentration of enzyme. binding equation -> formation of ES complex must be an important step in the reaction
5) Km is defined as the concentration of substrate at which V0 = Vmax
 Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

The catalysis exerted by an

enzyme can often be described This behavior indicates that for
with the equation: most enzymes their action can
Vmax [ S ] be described in two steps:
V0
Km ! [S ]
1) Relatively fast reversible binding
At low [S], [S]<<Km and then of E and S to form ES complex
most of the enzyme is free. k1
addition of more S increases the ###
E ! S %#$ ES
rate linearly (as a first order k"1

reaction): V ~ Vmax [ S ] 2) A slower step (limits the overall

0
Km rate of the reaction) that involves
the reversible breaking of the ES
At high [S], [S] >>> Km and then most of the enzyme is already bound to S (forming complex into E and P
the ES complex). Addition of more susbtrate does not change V0 significantly
k2
because V0 is already close to Vmax: V ~ V ##
ES %#$E ! P
#
0 max k2

Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

The behavior of enzyme reactions can be described quantitatively with the

At very early times after mixing
Michaelis-Menten treatment:
S and E there is fast formation of
ES complex. This phase is called k1 k2
pre-steady-state kinetics. ###
E ! S %#$ ES %##
##$ E ! P
k"1 k"2

Because the rate is measured at early times, [P] ~ 0 so that the back-reaction for
At slightly later times (but still early
the second step can be ignored
in terms of the amount of S converted
into P, or still under [S] ~ [S]T) the k1
concentration of ES reaches an almost ##
E ! S %# $ ES ##
#
k2
$E ! P
k"1
constant value by compensation
between the speed of its formation and
Because the second step is rate limiting, V0 is determined by the breakdown
the speed of its degradation. This phase
rate of the ES complex
is called steady-state kinetics

V0 k2 [ ES ]
 Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

The total amount of enzyme equals the amount of free enzyme plus the amount of The steady state approximation indicates that the concentration of the ES complex
ES complex: is constant at early times of the reaction

[ E ]T [ E ] ! [ ES ] d [ ES ]
0
The rate of formation of the ES complex is described as: dt
The rate of formation of the ES complex is simplified to:
d [ ES ]
k1[ E ][ S ] " k"1[ ES ] " k2 [ ES ]
dt 0 k1 &[ E ]T " [ ES ]' [ S ] " & k"1 ! k2 ' [ ES ]
Substituting and rearranging: Solving for [ES] we obtain:
d [ ES ] k1[ E ]T [ S ]
k1 &[ E ]T " [ ES ]' [ S ] " & k"1 ! k2 ' [ ES ] [ ES ]
dt k1[ S ] ! k"1 ! k2

Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

Dividing numerator and denominator by k1 What is the physical meaning of Km?

[ E ]T [ S ] If we solve for V0 = (1/2)Vmax: Vmax Vmax [ S ]

[ ES ]
[ S ] ! & k"1 ! k2 ' k1 2 [S ] ! Km
[ E ]T [ S ] 1 [S ]
By defining Km as (k-1+k2)/kl: [ ES ] 2 Km ! [S ]
[S ] ! Km
Because V0 = k2[ES]: K m ! [ S ] 2[ S ]
k2 [ E ]T [ S ] Km [ S ] when Vo &1 2 'Vmax
V0 Michaelis-Menten Equation
[S ] ! Km Km is the concentration of substrate at which V0 equals half Vmax

k2[E]T is the rate when all Vmax [ S ] if k-1>>>k2 (k2 rate limiting step) then Km approximates the dissociation constant
the enzyme is in the form V0 of the ES complex (k-1/k1).
of ES complex (Vmax)
[S ] ! Km if k-1<<<k2 (k=1 rate limiting step) then Km approximates (k2/k1).
 Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

Km values for various enzymes:

What is the physical meaning of Vmax?

Vmax kcat [ E ]T

Vmax is the product of the catalytic rate constant (or turnover number) by the
total concentration of enzyme in the assay. Therefore, Vmax depends on the
conditions of the assay. The important parameter is kcat, which is the same
for a given reaction at standard conditions.

kcat has units of s-1

The Km of an enzyme is normally close to the concentration of substrate in

physiological conditions to maximize the efficiency of the enzyme

Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

kcat values for various enzymes: Different enzymes might have similar efficiencies but very different
Km and kcat values.

The best parameter for comparing activities of various enzymes or various substrates
of the same enzyme is the Specificity Constant, which is defined as the rate
constant for conversion of E+S into E+P:

kcat
When [ S ] ((( K m ##
$V0 [ E ]T [ S ]
Km
kcat/Km is the specificity constant. The higher this parameter the more efficient
the enzyme (has units of a second order reaction: M-1s-1).

The specificity constant for an enzyme has an upper value of ~109 M-1s-1
determined by the rate of diffusion of the E and S molecules in aqueous solution.
The kcat varies widely from enzyme to enzyme and typically reflects an specificity constant near the diffusion-control value means that every time that
the specific rate of production needed for each particular product a molecule of E collides with one of S it catalizes its conversion into P.
 Enzymes 2: Enzyme Kinetics Enzymes 2: Enzyme Kinetics

Some enzymes for which the specificity constant is near Diffusion-Control

The quantitative analysis of enzyme kinetics is facilitated by linearizing the
Michaelis-Menten equation:

Vmax [ S ]
Vo
[S ] ! Km
1 Km ! [S ]
Vo Vmax [ S ]
1 Km 1
! Lineweaver-Burk Equation
Vo Vmax [ S ] Vmax
If the Km is very small (reflects very low [S] in vivo), then kcat cannot be too high,
While kcat can be very high for S that are abundant (high Km).

Enzymes 2: Enzyme Kinetics

The quantitative analysis of enzyme kinetics is facilitated by linearizing the
Michaelis-Menten equation:
Lineweaver-Burk Plot

Vmax [ S ]
Vo
[S ] ! Km
1 Km ! [S ]
Vo Vmax [ S ]
1 Km 1
!
Vo Vmax [ S ] Vmax
 17/08/2018

Immobilized Enzymes:

- Advantages of enzymes as industrial

catalyst OVER conventional catalyst.
- Advantages of immobilized enzymes
OVER soluble enzymes.
- Current and potential applications of
immobilized enzymes in industry.

1 2

Enzyme Reaction Process

3 4

Conventional Catalyst Enzyme Catalyst

Substances that increase or Proteins that increase rate of
Function decrease the rate of a chemical chemical reactions converting
reaction but remain unchanged. substrate into product.
Molecular Low molecular weight High molecular weight globular
weight compounds. proteins.
There are two types of catalysts There are two types of
Types – positive and negative enzymes - activation enzymes
catalysts. and inhibitory enzymes.
Catalysts are simple inorganic Enzymes are complex
Nature
molecules. proteins.
Alternate Organic catalyst or bio
Inorganic catalyst.
terms catalyst.
Reaction
Typically slower Several times faster
rates
They are not specific and Enzymes are highly specific
Specificity therefore end up producing producing large amount of
residues with errors good residues
Mild conditions, physiological
Conditions High temp, pressure
pH and temperature
C-C and C-
absent present
H bonds 5 6
Example vanadium oxide amylase, lipase

1
 17/08/2018

Enzyme Immobilization???? Enzyme Immobilization????

To restrict enzyme Immobilized enzymes are

mobility in a fixed ATTACHED
space. to an insoluble support medium or
enclosed by the support medium
which is also known as a CARRIER

In some cases, the enzymes molecules are

cross-linked to each other so that their
7
movement is restricted but their catalytic 8
activities are retained.

Need for Immobilizaton

continuous use of bio-catalyst
ECONOMICAL
is possible
separation of bio-catalyst &
CONVENIENCE product is much easier than
conventional batch process

Immobilized enzymes
typically have greater thermal
STABILITY & operational stability than
the soluble form of the
9 enzyme 10

11 12

2
 17/08/2018

Methods for immobilization enzyme Carrier-binding Method

 The oldest immobilization

method.
 Carriers (water insoluble)
such as polysaccharide
derivatives, synthetic
polymers, and porous glass,
etc.

13 14

Cross-linking Method Entrapping Method

 Based on confining enzymes in the lattice of a polymer matrix or enclosing
 Based on the formation of chemical bonds, but enzymes in semipermeable membranes.
 A chemical polymerization reaction e.g. collagen, gelatin, cellulose etc.
water-insoluble carriers are not used in this
method.
 Reagents such as glutaldehyde,
bisdiazobenzidine, and hexamethylene diisocyanate,
etc.

15 16

Preparation & Characteristics of

immobilized enzyme

Preparation &
Characteristics
of immobilized
enzyme

17 18

3
 17/08/2018

The properties of immobilized

enzymes

19 20

The properties of immobilized Characterization of immobilized

enzymes enzymes

scheme of a particle with immobilized

21
biocatalyst 22

23 24

4
 17/08/2018

25 26

27