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H.K.U.S.T
CENG 361
INTRODUCTION TO BIOCHEMICAL ENGINEERING AND
BIOPROCESSSING
BIOCHEMICAL ENGINEERING
PRODUCTS
LECTURE SLIDES PRESENTED
TO BIEN STUDENTS
PREPARED BY C.K. YEUNG
1
References:
• Atkinson, B., and M. Ferda, Biochemical Engineering and Biotechnology
Handbook, 2nd ed., Stockton Press, New York, NY (1991).
• Bailey, J. E., and D. F. Ollis, Biochemical Engineering Fundamentals, 2nd
ed., McGraw Hill, New York, NY (1986).
• Blanch, H. W., and D. S. Clark, Biochemical Engineering, Marcel Dekker,
New York, NY (1996).
• Buerk, D. G., Biosensors: Theory and Applications, Technomic Publishing
Company, Inc., Lancaster, PA (1993).
• Gebelein, C. G., ed., Biotechnological Polymers, Technomic Publishing
Company, Inc., Lancaster, PA (1993).
• Hall, E. A. H., Biosensors, Prentice Hall, Inc., Englewood Cliffs, NJ
(1991).
• Harsanyi, G., Sensors in Biomedical Applications: Fundamentals,
Technology and Applications, Chapter 7, Technomic Publishing Company,
Inc., Lancaster, PA (2000).
• Ouellette, R. P., and P. N. Cheremisinoff, Applications of Biotechnology,
Technomic Publishing Company, Inc., Lancaster, PA (1985).
• Rho, J. P., and S. G. Louie, Handbook of Pharmaceutical Biotechnology,
2
Haworth Press, Inc., Binghamton, NY (2003).
Major classes of bioproducts, such as chemical,
biochemical,
biopharmaceutical
and
bioengineered products will be introduced.
The significant impacts of these bioproducts will also
be discussed. This course covers the processes and
production of certain bioproducts and the methods
that can be used for their separation, purification
and identification. Some current approaches to the
bioproduct productions and applications including
recombinant
DNA
technology,
cell/tissue
engineering, product forms and bio-devices will
also be introduced.
3
Major Classes of Bioproducts
(Products derived from bio-sources
or used in bio-applications)
1)Basic Chemicals
2)Biochemicals
3)Biopharmaceuticals
4)Engineered Bioproducts
4
1. Basic Chemicals
Organic Acids (Citric Acid, Lactic Acid)
Alcohols (1,3 Propanediol)
Amino Acids (Glutamic Acid, Lysine)
2. Biochemicals
Enzymes (Proteolytic Enzymes)
Surfactants (Lecithin, Esters)
5
3. Biopharmaceuticals
Antibiotics (Penicillin)
Monoclonal/Polyclonal Antibodies
Hormones (Growth Hormones)
Vaccines (Hep B Vaccine)
Therapeutic Proteins (tPA)
4. Engineered Products
Bio-devices (Bio-devices, Microorganisms
DNA microarray chips, tissue/cell based)
6
Major Classes of Bioproducts
(Products derived from bio-sources
or used in bio-applications)
1)Basic Chemicals
2)Biochemicals
3)Biopharmaceuticals
4)Engineered Bioproducts
7
1. Basic Chemicals
Organic Acids
Citric Acid
2-hydroxy-1,2,3-propane-tricarboxylic or betahydroxytricarballylic acid.
As part of the tricarboxylic acid (TCA) cycle
Citrate synthase catalyses the reaction between
acetyl-CoA and oxaloacatate to form citric acid
8
The TCA cycle
9
(Citric Acid)
HO2CCH2C(OH)(CO2H)CH2CO2H, an organic
carboxylic acid containing three carboxyl groups;
Citric acid, anhydrous, crystallizes from hot
aqueous solutions as colorless translucent
crystals or white crystalline powder.
Citric acid is deliquescent in moist air and is
optically inactive.
10
(Citric Acid)
It is a solid at room temperature,
Melts at 153°C,
Taste of various fruits in which it occurs, e.g.,
lemons, limes, oranges,
Citric acid loses water at 175 °C to form aconitic
acid, HOOCCH=C(COOH) (CH2COOH), which
loses carbon dioxide to yield citraconic
anhydride
11
(Citric Acid)
Itaconic anhydride rearranges to citraconic
anhydride (see Fig) or adds water to form
itaconic acid , (HOOCCH2 ) (HOOC)C=CH2
Add water to Citraconic anhydride: gives
citraconic acid, cis-HOOCCH=C(CH3) (COOH).
Evaporation of a citraconic acid solution in the
presence of nitric acid yields mesaconic acid, the
trans isomer of citraconic acid.
12
+H2O
+H2O
Itaconic Acid
Citraconic Acid
13
Citric Acid – Source and Production
Can be extracted from the juice of citrus fruits by
adding calcium oxide (lime) to form calcium
citrate,
Precipitate can be collected by filtration,
Citric acid can be recovered from its calcium salt
by adding sulfuric acid.
It is obtained also by fermentation of glucose with
the aid of the mold Aspergillus
14
The most economical method for producing citric
acid since the 1930s has been fermentation, which
employs a strain of Aspergillus niger to convert sugar
to citric acid. Both surface fermentation and
submerged fermentation have been used.
15
Citric Acid – Surface Fermentation (1)
A. niger is grown on a liquid substrate in pans
stacked vertically in a chamber
The chamber and pans are sterilised either
before or after introduction of the substrate
The pans are filled manually or automatically.
The chamber is warmed by the introduction of
moist, sterile air at a controlled temperature.
16
Citric Acid – Surface Fermentation (2)
The liquid and the surface microorganisms are
removed manually or automatically from the pans
The pans are cleaned before the next batch is
introduced.
The substrate for the fermentation is a
carbohydrate, usually a sugar, such as raw beet,
refined beet, or cane sugars, or a syrup.
17
Citric Acid – Surface Fermentation (3)
Glucose syrups can be prepared from wheat,
corn, potato, or other starch.
The sugar content of the syrup can vary from
about 10 to 25 wt %.
Certain inorganic nutrients, such as (1)
ammonium nitrate, (2) potassium phosphate, (3)
magnesium sulfate, (4) zinc sulfate, and (5)
potassium ferrocyanide, are added.
18
Citric Acid – Surface Fermentation (4)
The pH is adjusted to between 3 and 7,
depending on the carbohydrate source.
Sterilisation may be batchwise or continuous;
the latter uses less energy and is usually faster.
After sterilisation, the temperature is adjusted
as required.
19
Citric Acid – Surface Fermentation (5)
The surface of the sterile substrate in the pans is
inoculated with A. niger spores, which germinate
and cover the surface of the liquid with a matt of
mold.
After two to three days the surface is completely
covered and citric acid production begins,
continuing at almost a constant rate until 80 –
90 % of the sugar is consumed.
Fermentation then continues more slowly for an
additional six to ten days.
20
Citric Acid – Surface Fermentation (6)
The theoretical yield from 100 kg of sucrose is
123 kg of citric acid monohydrate or 112 kg of
anhydrous acid.
However, the A. niger uses some sugar for
growth and respiration, and the actual yield
varies between 57 and 77 % of theoretical
value, depending on such factors as:
(1) Substrate purity,
(2) Particular strain of organism, and
(3) Control of fermentation
21
Citric Acid – Submerged Fermentation (1)
Submerged fermentation is similar to surface
fermentation, but takes place in large
fermentation tanks.
This method is used more frequently because
labour costs are lower with large tanks than with
small pans;
Equipment costs are also lower.
22
Citric Acid – Submerged Fermentation (2)
The fermentation vessel can be short and wide
or tall and narrow, and equipped with mixing
devices, such as top-entering or side-entering
agitators of the turbine or propeller type.
Agitation can be increased by use of a draft
tube, a re-circulation loop, or a nozzle through
which air and re-circulated substrate is pumped.
23
Citric Acid – Submerged Fermentation (3)
Spargers (agitation by means of compressed air)
located at the bottom of the vessel or under the
stirrer supply air, which may be enriched with
oxygen.
Oxygen is usually recovered from the exhaust
gas.
The air is supplied by a compressor and passes
through a sterile filter; if necessary, the air is
cooled.
24
Citric Acid – Submerged Fermentation (4)
Because the process is exothermic, the vessel
must be equipped with heat-exchange
surfaces, which can be the outside walls or
internal coils.
Ports are provided for introducing substrate,
inoculum, and steam or other sterilising agents;
sampling and exhaust ports are also provided.
25
Citric Acid – Submerged Fermentation (5)
The substrate is prepared in a separate tank
and its pH adjusted;
The micronutrients may be added to this
tank or directly to the fermenter.
The substrate is sterilised by a batchwise or,
more commonly, by a continuous operation.
26
Citric Acid – Submerged Fermentation (6)
The fermenter is sterilised,
substrate, and inoculated.
charged
with
Fermentation requires 3 –14 days.
After it is completed, the air supply is stopped to
prevent the microorganisms from consuming the
citric acid.
27
Citric Acid – Recovery (1)
The citric acid broth from the surface or
submerged fermentation processes must be
purified.
First, biological solids usually are removed by
filtration using a rotary vacuum filter or the
more recent belt-press filter, or by centrifugation.
The solids are washed to improve recovery
of citric acid.
28
Citric Acid – Recovery (2)
The dissolved citric acid must then be
separated from residual sugars, proteins
generated by the fermentation, and other
soluble impurities.
This has traditionally been accomplished by
precipitation and crystallisation.
Addition of lime precipitates calcium citrate,
which is filtered and stirred in dilute sulfuric
acid to form a precipitate of calcium sulfate;
filtration yields a purified citric acid solution
29
Dissolved Citric Acid + Lime

calcium citrate (ppt)

filtered and stirred in dilute sulfuric acid

calcium sulfate (ppt)

filtration

purified citric acid solution
30
Citric Acid – Recovery (3)
Control of pH and temperature in these
operations helps to optimise the results.
Citric acid is then crystallised from solution
and recrystallised from water;
The mother liquors are recycled to remove
accumulated impurities
31
Citric Acid – Its use
Can be obtained synthetically from acetone or
glycerol.
Citric acid is used in soft drinks (45%) and in
laxatives and cathartics.
Its salts, the citrates, have many uses, e.g., ferric
ammonium citrate is used in making blueprint
paper.
Sour salt, used in cooking, is citric acid
32
Citric Acid
• Reference
– The Merck Index, 11th ed., Merck & Co., Rahway, N.J. 1989.
– R. C. Weast, CRC Handbook of Chemistry and Physics, 69th ed.,
CRC Press, Boca Raton, Fla., 1988 CRC Handbook of
Chemistry and Physics, 1989, p. 163.
– A. Seidell, Solubilities of Inorganic and Organic Compounds, 3rd
ed., Vol. 2, D. Van Nostrand Co., Inc., New York, 1941, 427–429.
– Ethyl Corp., DE-OS 2 240 723, 1972.
– M. Rohr, C. P. Kubicek, J. Kominek: “Citric Acid” in H. Dellweg
(ed.): Biotechnology Microbiology Products, Biomass, and
Primary Products, vol. 3, Verlag Chemie, Weinheim 1983,
pp. 456 – 465.
– G. T. Austin, Shreve's Chemical Process Industries, 5th ed.,
McGraw-Hill Book Co., Inc., New York, 1984.
33
Lactic acid
It was first discovered in 1780 by the Swedish
chemist Scheele.
CH3CHOHCO2H, is the most widely occurring
hydroxycarboxylic acid
A colorless liquid organic acid.
Miscible with water or ethanol.
34
(Lactic acid)
Lactic acid is a naturally occurring organic acid
that can be produced by fermentation or
chemical synthesis.
Lactic acid is also a principal metabolic
intermediate in most living organisms, from
anaerobic prokaryotes to humans
Anhydrous lactic acid is a white, crystalline solid
with a low melting point. Generally, it is available
as a dilute or concentrated aqueous solution.
35
Lactic acid – Its use (1)
A fermentation product of lactose (milk sugar)
Is produced in muscles during intense activity.
Calcium lactate, a soluble lactic acid salt, is
used as a source of calcium in the die
Present in sour milk, yogurt, and cottage cheese
(in situ microbial fermentation).
36
Lactic acid – Its use (2)
The protein in milk is coagulated (curdled = go
bad!) by lactic acid.
Lactic acid is produced commercially for use in
pharmaceuticals and foods, in leather tanning
and textile dyeing, and in making plastics,
solvents, inks, and lacquers (paint / natural
varnishes a solution of cellulose derivative).
37
Lactic acid – Production (1)
Although it can be prepared by chemical
synthesis, production of lactic acid by
fermentation
of
glucose
and
other
substances is a less expensive method.
Chemically, lactic acid occurs as two optical
isomers, a dextro and a levo form; only the levo
form takes part in animal metabolism.
The lactic acid of commerce is usually an
optically inactive racemic mixture of the two
isomers.
38
• Lactic acid is the simplest hydroxy acid that is optically
active. L-Lactic acid (1) occurs naturally in blood and in
many fermentation products. The chemically produced
lactic acid is a racemic mixture and some fermentations
also produce the racemic mixture or an enantiomeric
excess of D-lactic acid (2)
39
Lactic acid – Production (2)
The commercial process is based on lactonitrile
which used to be a by-product of acrylonitrile
synthesis.
It involves base-catalysed addition of hydrogen
cyanide to acetaldehyde to produce
lactonitrile
This is a liquid-phase reaction and occurs at
atmospheric pressures
40
Lactic acid – Production (3)
The crude lactonitrile is then recovered and
purified by distillation and is hydrolysed to
lactic
acid
using
either
concentrated
hydrochloric or sulphuric acid, producing the
corresponding ammonium salt as a by-product.
This crude lactic acid is esterified with
methanol, producing methyl lactate (see next slide)
41
Lactic acid – Production (4)
The latter is recovered and purified by distillation
and hydrolysed by water under acid catalysts to
produce lactic acid, which is further concentrated,
purified, and shipped under different product
classifications, and methanol, which is recycled.
42
Lactic acid – Fermentation (1)
The existing commercial production processes
use
homolactic
organisms
such
as
Lactobacillus delbrueckii, L. bulgaricus, and
L. leichmanii.
A wide variety of carbohydrate sources, eg,
molasses, corn syrup, whey, dextrose, and cane
or beet sugar, can be used.
Other complex nutrients required by the
organisms are provided by corn steep liquor,
yeast extract, soy hydrolysate, etc.
43
Lactic acid – Fermentation (2)
Excess calcium carbonate/hydroxide is
added to the fermenters to neutralise the acid
produced and produce a calcium salt of the acid
in the broth.
The fermentation is conducted batchwise,
taking 4–6 days to complete, and lactate
yields of approximately 90 wt% from a dextrose
equivalent of carbohydrate are obtained.
44
Lactic acid – Fermentation (3)
It is usually desired to keep the calcium lactate
in solution so that it can be easily separated
from the cell biomass and other insolubles,
This limits the concentration of carbohydrates
that can be fed in the fermentation and the
concentration lactate in the fermentation broth,
which is usually around 10 wt%.
45
Lactic acid – Fermentation (4)
The calcium lactate-containing broth is filtered to
remove cells, carbon-treated, evaporated, and
acidified with sulfuric acid to convert the salt
into lactic acid and insoluble calcium sulfate,
which is removed by filtration (See Figure)
The filtrate is further purified by carbon columns
and ion exchange and evaporated to produce
technical- and food-grade lactic acid, but not a
heat-stable product, which is required for the
stearoyl lactylates, polymers, and other valueadded applications.
46
Lactic acid – Fermentation (5)
47
48
Lactic Acid
• References
– C. H. Holten, A. Muller, and D. Rehbinder, Lactic Acid,
International Research Association, Verlag Chemie,
Copenhagen, Denmark, 1971.
– S. C. Prescott and C. G. Dunn, Industrial Microbiology, 3rd ed.,
McGraw-Hill Book Co., Inc., New York, 1959.
– R. C. Schulz and J. Schwaab, Makromol. Chem. 87, 90–102
(1965).
– Biomass Process Handbook, Technical Insights, Inc., Fort Lee,
NJ., 1982, 96–103.
49
Alcohols
1,3 Propanediol (PDO) – Properties
1,3-Propanediol,
trimethylene
glycol,
HOCH2CH2CH2OH, is a clear, colorless, odorless
liquid that is miscible with water, alcohols, ethers, and
formamide.
It is sparingly soluble in benzene and chloroform.
The chemical properties of 1,3-propanediol are typical
of alcohols.
50
1,3 Propanediol (2)
It reacts with isocyanates and acid chlorides to
yield urethanes and esters, respectively.
Unlike 1,2-propanediol, 1,3-propanediol has two
primary hydroxyl groups with equivalent
reactivity.
51
1,3 Propanediol (3)
1,3-Propanediol readily forms ethers. 3,3Dihydroxydipropyl ether forms upon continued
reflux of the diol.
1,3-Propanediol reacts with aldehydes and
ketones, often in the presence of acidic
catalysts, to form 1,3-dioxanes:
52
1,3 Propanediol (4)
Hydrolysis is carried out under weakly acidic
conditions in water containing initially ca. 20 %
acrolein.
Higher concentrations of acrolein tend to lead to
greater amounts of undesired byproducts as a
result of reaction between acrolein (a colorless
irritant pungent liquid aldehyde C3H4O used chiefly in organic
synthesis) and hydroxy-propanal)
53
1,3 Propanediol (5)
3-Hydroxypropanal can be hydrogenated in the
aqueous phase directly; however, the preferred
technique is to extract the aldehyde into an organic
solvent — particularly 2-methylpropanol — and then
hydrogenate the aldehyde to yield the diol.
Hydrogenation is conducted with Raney nickel under
pressure in the aqueous phase and with nickelsupported catalysts at 2 – 4 MPa and 110 –150 °C in
the organic phase.
The diol is subsequently separated from solvent and
water by distillation.
54
1,3 Propanediol (6)
The yield of desired product by this route is
approximately 45 %.
Hydroformylation of ethylene oxide followed by
hydrogenation yields 1,3-propanediol in good
yield (92 %), but a high catalyst concentration
and a very large excess of solvent render the
process uneconomical.
55
1,3 Propanediol (7)
More recently, hydroformylation of ethylene oxide
directly to 1,3-propanediol with a rhodium –
phosphine catalyst system has been disclosed.
The reaction of ethylene with formaldehyde and
carboxylic
acids
has
also
not
been
commericialised because of low selectivity
56
1,3-Propanediol
• Reference
– Ullmann, 4th ed., 19, 425 – 432
– J. L. Mateo, O. RuizMurillo, R. Sastre, An. Quim. Ser C 80 (1984)
no. 2, 178
– L. F. Lapuka et al., Khim Geterotsikl. Soedin. 1981, no. 9, 1182;
Chem. Abstr. 95 (1981) 20 039 e.
– R. W. Lenz: Organic Chemistry of Synthetic High Polymers,
Interscience, New York 1967, p. 93.
– Shell Oil Company, US 3 463 910, 1970 (C. N. Smith, G. N.
Schrauzer, K. F. Koetitz, R. J. Windgassen).
– Hoechst Celanese, US 4 873 378, 1989 (M. Murphy et al.).
– National Distillers and Chemical Corp., US 4 322 355, 1980 (D.
Horvitz, W. D. Bargh).
57
Amino Acids
•
•
1)
2)
3)
4)
Amino acids are the building
blocks of proteins
There is
–COOH, which is a carboxyl
group (acidic)
-NH2, which is an amino
group (basic)
an –H hydrogen
a residue R which varies
depending on the amino acid
58
Amino Acids (cont)
•
All 20 essential amino acids have this same structure
but their side chain groups ‘R’ may vary in size,
shape, charge, hydrophobicity and reactivity
1)
2)
3)
4)
–COOH, which is a carboxyl group (acidic)
-NH2, which is an amino group (basic)
a –H hydrogen
a residue R which varies depending on the amino
acid
59
AA side chains are sorted into groups
The side chain is an aliphatic group (hydrophobic)
•
Glycine (Gly)
•
Alanine (Ala)
•
Valine (Val)
•
Leucine (Leu)
•
Isoleucine (Ile)
The side chain is an organic acid (negatively charged)
•
Aspartic Acid (Asp)
•
Glutamic Acid (Glu)
The side chain contains a sulphur (Hydrophobic)
•
Methionine (Met)
•
Cysteine (Cys)
The side chain is an alcohol
•
Serine (Ser)
•
Threonine (Thr)
•
Tyrosine (Tyr)
The side chain is an organic base (hydrophilic)
•
Arginine (Arg)
•
Lysine (Lys)
•
Histidine (His)
The side chain is aromatic (very hydrophobic)
•
Phenylalanine (Phe)
•
Tryptophan (Trp)
The side chain is an imine
•
Proline (Pro)
60
Glutamic Acid:
An amino acid, HOOCCH2CH2CH(NH2)COOH,
Obtained by hydrolysis from wheat gluten and
sugar-beet residues
Used commercially chiefly in the form of its sodium
salt (MSG) to intensify the flavor of meat or other
food
Like aspartic acid, glutamic acid has an acidic
carboxyl group on its side chain which can serve as
both an acceptor and a donor of ammonia, a
61
compound toxic to the body.
Lysine:
Organic compound, one of the 20 AAs commonly
found in animal proteins.
Only the l-stereoisomer appears in mammalian
protein.
The human body cannot synthesise it from simpler
metabolites.
Young adults need about 23 mg of this amino acid
per day per kilogram (10 mg per lb) of body weight.
62
2,6-Diaminohexanoic acid C6H14N2O2
• Has a net positive charge at physiological pH values
making it one of the three basic amino acids.
• This polar amino acid is commonly found on the
surfaces of proteins and enzymes, and sometimes
appears in the active site. Sources of lysine include
meats, fish, poultry, and dairy products.
63
Lysine is found in particularly low concentrations
in the proteins of cereals; wheat gluten, for
example, is relatively poor in lysine.
This deficiency in lysine is the reason for the
failure of diets in some parts of the world that
employ cereal protein as a sole source of
essential amino acids to support growth in
children and general well-being in adults.
- kwashiorkor
Attempts to develop lysine-rich corn have been
partly successful.
64
Once lysine is incorporated into protein, its basic
side chain often provides a positive electrical
charge to the protein, thereby aiding its solubility in
water.
Its side chain has also been implicated in the
binding of several coenzymes (pyridoxal
phosphate, lipoic acid, and biotin) to enzymes. It
also plays an important role in the functioning of
histones.
The amino acid was first isolated from casein (milk
protein) in 1889
65
Production
66
Extraction of Amino Acid
67
Production by Fermentation (1)
The most potent microorganisms to overproduce
lysine
are
mutants
derived
from
Corynebacterium glutamicum, a gram-positive
bacterium.
Mainly auxotrophic and regulatory mutants of this
bacterium have been developed
Cell fusion with the method of protoplast (a plant cell
with its cell wall removed) fusion has been applied for
breeding of industrial microorganisms.
68
Production by Fermentation (2)
This technique allows the combination of
positive characteristics of different strains such
as high selectivity and high productivity.
In fermentation with media of inhibitory osmotic
stress the sugar consumption rate and lysine
production rate of some mutants can be
stimulated by the addition of glycine.
69
Production by Fermentation (3)
In fed-batch culture and under appropriate
conditions the favorable mutants for lysine
production are able to reach final concentration
of about 120 g/L lysine.
Fermentation processes are performed in big
tanks up to 500 m3 size.
70
Production by Fermentation (4)
The conventional route of lysine downstream
processing is characterised by:
Removal of the bacterial cells from
fermentation broth by separation or ultrafiltration
Absorbing and then collecting lysine in an
ion exchange step
Crystallising or spray drying of lysine as llysine hydrochloride
71
Lysine
• References
– http://www.adhd-becalmd.com/neurotransmitters/8/l-lysineamino-acid.html
– M. Karasawa, O. Tosaka, S. Ikeda, H. Yoshi, Agric. Biol. Chem.
50 (1986) 339 – 346.
– Kyowa Hakko, US 4 623 623, 1986 (T. Nakanashi et al.).
– Y.-C. Liu, W.-T. Wu, J.-H. Tsao, Bioprocess. Eng. 9 (1993) 135 –
139.
72
Major Classes of Bioproducts
(Products derived from bio-sources
or used in bio-applications)
1)Basic Chemicals
2)Biochemicals
3)Biopharmaceuticals
4)Engineered Bioproducts
73
2. Biochemicals
Enzymes
The purpose of an enzyme in a cell is to allow the cell to
carry out chemical reactions very quickly.
Enzymes are made from amino acids, and they are
proteins. When an enzyme is formed, it is made by
stringing together between 100 and 1,000 amino acids in a
very specific and unique order.
The chain of amino acids then folds into a unique shape.
That shape allows the enzyme to carry out specific
chemical reactions -- an enzyme acts as a very efficient
catalyst for a specific chemical reaction.
74
Proteolytic Enzymes
1. Renin
(secreted by the kidneys that is involved in the
release of angiotensin)
2. Trypsin
(enzyme of the pancreatic juice, capable of
converting proteins into peptone)
75
Proteolytic Enzymes (cont).
3. Pepsin
Enzyme produced in the mucosal lining of the
stomach that acts to degrade protein.
Pepsin is one of three principal protein-degrading,
or proteolytic, enzymes in the GIT, the other two
being chymotrypsin and trypsin.
During the process of digestion, these enzymes,
break down dietary proteins to their components,
i.e., peptides and AAs.
76
Proteolytic Enzymes (cont).
3. Pepsin (cont)
Pepsin is synthesised in an inactive form by the
stomach lining; hydrochloric acid, also produced
by the gastric mucosa, is necessary to convert the
inactive enzyme and to maintain the optimum
acidity (pH 1–3) for pepsin function.
Pepsin and other proteolytic enzymes are used in
the laboratory analysis of various proteins; pepsin
is also used in the preparation of cheese and other
protein-containing foods.
77
Proteolytic Enzymes (cont).
4. Papain
A proteolytic enzyme found in the fruit of the
papaya tree
A commercial preparation of this used as a meat
tenderiser and in medicine as a digestant
78
Proteolytic Enzymes (cont).
5. Chymotrypsin
Found in pancreatic juice
Catalyses the hydrolysis of proteins into polypeptides and amino acids
6. Subtilisin
Produced by the bacterium Bacillus subtilis,
used as an active ingredient in detergents and
also in research to help reveal protein
structure.
79
Proteolytic Enzymes (cont).
7. Fibrinolysin (also known as plasmin)
Formed in the blood from plasminogen, that
causes the breakdown of the fibrin in blood
clots
8. Cathepsin
Intracellular proteolytic enzymes, occurring in
animal tissue, esp. the liver, spleen, kidneys,
and intestine, that catalyze autolysis in certain
pathological conditions and after death.
80
Surfactants
Any substance that when dissolved in water or an
aqueous solution reduces its surface tension or the
interfacial tension between it and another liquid
1. Lecithin
Group of phospholipids, occurring in animal and
plant tissues and egg yolk, composed of units of
choline, phosphoric acid, fatty acids, and glycerol.
A commercial form of this substance, obtained
chiefly from soybeans, corn, and egg yolk, used in
foods, cosmetics, and inks.
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Any substance that when dissolved in water (form
colloidal solutions in water) or an aqueous solution
reduces its surface tension or the interfacial
tension between it and another liquid, have
emulsifying, wetting, and antioxidant properties
2. Esters
Any one of a group of organic compounds with
general formula RCO2R' (where R and R' are alkyl
groups or aryl groups) that are formed by the
reaction between an alcohol and an acid.
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2. Esters (cont).
When ethanol and acetic acid react, ethyl acetate
(an ester) and water are formed; the reaction is
called esterification.
Ethyl acetate is used as a solvent. Methyl acetate,
formed by the reaction between methanol and
acetic acid, is a sweet-smelling liquid used in
making perfumes, extracts, and lacquers.
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2. Esters (cont).
Esters react with water (hydrolysis) under basic
conditions to form an alcohol and an acid.
When heated with a hydroxide certain esters
decompose to yield soap and glycerin; the
process is called saponification.
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2. Esters (cont).
Common fats and oils are mixtures of various esters,
such as stearin, palmitin, and linolein, formed from
the alcohol glycerol and fatty acids.
Naturally occurring esters of organic acids in fruits
and flowers give them their distinctive odors.
Esters perform important functions in the animal
body; e.g., the ester acetylcholine is a chemical
transmitter of nerve stimuli.
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Major Classes of Bioproducts
(Products derived from bio-sources
or used in bio-applications)
1)Basic Chemicals
2)Biochemicals
3)Biopharmaceuticals
4)Engineered Bioproducts
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3. Biopharmaceuticals
Antibiotics
Eg. Penicillins
Several antibiotics of low toxicity
Produced naturally by molds of the genus
Penicillium and also semi-synthetically
Having a bactericidal action on many susceptible
Gram-positive or Gram-negative cocci and bacilli
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Antibiotics
Penicillins (cont)….
Includes: Cloxacillin, floxacillin, ampicillin, Penicillin
G and Penicillin V
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Antibodies
Methods of Obtaining Antibodies
•
Traditionally, antibodies to human gene products
have traditionally been obtained by repeatedly
injecting suitable animals (rodents, rabbits, cats
and dogs, goats etc) with a suitable immunogen.
2 types are commonly used:
1) Synthetic Peptides
2) Fusion Proteins
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How do you get antibodies in your
body?
The Original View (the Wrong View)
Each cell makes antibodies of only one kind
Stimulation of cell division and antibody synthesis occurs
after interaction of antigen with receptor antibodies at the
cell surface
The specificity of these antibodies is the same as that of
the antibodies produced by daughter cells.
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How do you get antibodies in your
body?
The Present View (the Right View) – Nobel Laureate,
Gerald Edelman
The binding of antigens induces clonal proliferation of
lymphoid cells
Molecular recognition of antigens occurs by selection
among clones of cells already committed to producing the
appropriate antibodies, each of different specificity
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Synthetic Peptides
The amino acid sequence is inspected and a
synthetic peptide (often 20-50 amino acids long) is
designed.
The idea is that when conjugated to a suitable
molecule, it will undergo conformational change.
This will then adopt a structure resembling the native
polypeptide.
Doesn’t really work! (It is difficult to generate suitably
specific antibodies)
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Fusion Proteins
To insert a suitable cDNA sequence into a modified
bacterial gene contained within an appropriate
expression cloning vector
The rationale is that a hybrid mRNA will be produced
which will be translated to give a fusion protein with
an N-terminal region derived from the bacterial gene
and the remainder derived from the inserted gene.
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Antibodies (Polyclonal)
If the animal system has responded, specific
antibodies should be secreted into the serum
The antibody-rich serum (antiserum) which is
collected contains a heterogeneous mixture of
antibodies, each produced by a different B
lymphocyte
The different antibodies recognise different parts
(Epitopes) of the immunogen (Polyclonal Antisera)
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Antibodies (Monoclonal)
An antibody that is mass produced in the laboratory
from a single clone and that recognizes only one
antigen.
Monoclonal antibodies are typically made by fusing
a normally short-lived, antibody-producing B cell to
a fast-growing cell, such as a cancer cell.
The resulting hybrid cell, or hybridoma (see next
series of slides for details), multiplies rapidly, creating
a clone that produces large quantities of the
antibody.
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Hybridoma
Because B cells have a limited life-span in culture, it
is preferrable to establish an immortal cell line of
antibody-producing cells
Hybridomas – ‘hybrid myeloma’ (are propagated as
individual clones, each can provide a permanent and
stable source of a single type of monoclonal
antibody (mAb)
(See Figure included)
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Hybridoma (cont).
Animals produce highly heterogeneous mixtures of
antibodies (Ab 1, 2 etc) secreted by different clones of
immunocytes (cells 1 ,2 etc).
This is recognised by the antigen
Hybrids between Spleen cells (spleenocytes) from
hyperimmune animals are fused with Myeloma cells
produce monoclonal antibodies directed against simple
antigenic determinants.
Once isolated, the hybrid clones can be grown in
unlimited quantity in vitro or can be grown as tumours in
recipient animals.
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Hybridoma (cont).
Upon fusion, cultures are grown in the so-called HAT
(Hypoxanthine, Aminopterin, Thymidine) selective
medium.
The myeolma cells
(hypoxanthine guanine
(HGPRT)).
are lacking an enzyme
phosphoribosyl transferase
These mutants are unable to survive in HAT
Aminopterin blocks the main biosynthesis for the
production of nuclei acids and the cells use the so-called
salvage pathway HGPRT and thymidine kinase.
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Hybridoma (cont).
The immunocytes from hyperimmune animals provide
the genetic material for the production of a specific
antibody and HGPRT, allowing the hybrid to grow in
HAT medium.
Non-fused parental myeloma will disappear in HAT
while non-fused immunocytes are over grown by the
hybrids.
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Genetically-Engineered Antibodies
DNA cutting and ligation technology could be
used to generate new antibodies including both
partially humanised antibodies and fully humanised
antibodies.
Transgenic mice have been engineered to contain
human immunoglobulin loci permitting the in vivo
production of fully human antibodies.
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Hormones
Secretary substance carried from one gland or
organ of the body via the bloodstream to more
or less specific tissues, where it exerts some
influence upon the metabolism of the target
tissue.
Produced and secreted by the endocrine glands
including the pituitary, thyroid, parathyroids,
adrenals, ovaries, testes, pancreatic islets, certain
portions of the gastrointestinal tract, and the
placenta, among the mammalian species.
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Hormones
The anterior pituitary include thyrotropin,
adrenocorticotropic hormone, the gonadotropic
hormones and growth hormone
The posterior pituitary secretes antidiuretic
hormone, prolactin, and oxytocin. The thyroids
secrete thyroxine and calcitonin, and the
parathyroids secrete parathyroid hormone.
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Hormones
The adrenal medulla secretes epinephrine and
norepinephrine while the cortex of the same gland
releases aldosterone, corticosterone, cortisol, and
cortisone.
The ovaries primarily secrete estrogen and
progesterone and the testes testosterone.
The adrenal cortex, ovaries, and testes in fact
produce at least small amounts of all of the steroid
hormones.
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Hormones
The islets of Langerhans in the pancreas
secrete insulin, glucagon, and somatostatin.
The kidneys also produce erythropoietin, which
produces erythrocytes (red blood cells)
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Human Growth Hormones
Growth hormone or somatotropin , glycoprotein
hormone released by the anterior pituitary gland
that is necessary for normal skeletal growth in
humans.
Evidence suggests that the secretion of human
growth hormone (HGH) is regulated by the
release of certain peptides by the hypothalamus of
the brain.
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Human Growth Hormones (cont).
One such substance, called somatostatin, has
been shown to inhibit the secretion of HGH.
HGH is known to act upon many aspects of
cellular metabolism, but its most obvious effect is
the stimulation of the growth of cartilage and
bone in children.
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Vaccines
Definition:
Any preparation used as a preventive inoculation
to confer immunity against a specific disease,
Usually employing an innocuous form of the
disease agent, as killed or weakened bacteria or
viruses, to stimulate antibody production
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Vaccines (Hepatitis)
Hep A
Infectious hepatitis, occurs sporadically or in epidemics,
the virus being present in feces and transmittable via
contaminated food or water.
Spreads by physical contact
The disease usually resolves on its own.
Exposed persons can be protected by injections of
gamma globulin.
A vaccine was made available in 1995 and is
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recommended for children at risk for the virus.
Hep B
Serum hepatitis, was commonly transmitted
through blood transfusions.
Intravenous-drug abusers remain a high-risk
group
Spread by sexual transmission and from mother
to baby at birth.
Some infected individuals, particularly children,
become chronic carriers of the virus.
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Hep B (Cont).
HepB can progress to chronic liver disease and is
associated with an increased risk of developing
liver cancer.
A vaccine, available since 1981, is recommended
for all infants and others at risk for the virus.
Alpha-interferon was approved as a treatment in
1992
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Hep C
Formerly called non-A, non-B hepatitis
Is also transmitted by contaminated blood
transfusions and by sharing of needles.
It is the most common form of chronic liver
disease in the US.
Many of those infected have no symptoms but
become carriers
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Hep C (Cont).
The virus may eventually cause liver damage.
Blood banks routinely screen for hepatitis C.
Alpha-interferon is used also to treat hepatitis
C, in combination with the drug ribavirin.
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Hep D
Delta hepatitis, affects only people with hepatitis
B
Those infected with both viruses tend to have
more severe symptoms
Hep E
Is spread by consuming feces-contaminated
food or water. It is common in Mexico, Africa,
and Asia and is especially serious in pregnant
women.
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Therapeutic Proteins
Proteins that are used as drug ingredients
Eg. porcine insulin, blood coagulation factors VIII
and IX (Christmas), pancreatin etc….
But…there is the risk of allergic (Immune) reaction
Rapid inactivation
Can’t be used repeatedly
Risk of infection (HIV, Hepatitis)
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Therapeutic Proteins (cont)
Gene technology aided the production of large
amount of protein drugs
tPAs such as hormones, growth factors and some
can act as biocatalysts or inhibitors
Huge impact on physiological processes
Examples:
-Interferon
(HepB
Vaccine),
Coagulation factors VIII (Haemophilia A), Interferon (chronic granulomatous disease) etc.
DNase (cystic fibrosis) etc…
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