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Title: H.K.U.S.T CENG 361 INTRODUCTION TO BIOCHEMICAL ENGINEERING AND BIOPROCESSSING


1
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

2
  • 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, Haworth Press,
    Inc., Binghamton, NY (2003).

3
Major classes of bioproducts, such as chemical,
biochemical, biopharmaceutical and bio-
engineered 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.
4
Major Classes of Bioproducts (Products derived
from bio-sources or used in bio-applications)
  • Basic Chemicals
  • Biochemicals
  • Biopharmaceuticals
  • Engineered Bioproducts

5
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)
6
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)
7
Major Classes of Bioproducts (Products derived
from bio-sources or used in bio-applications)
  • Basic Chemicals
  • Biochemicals
  • Biopharmaceuticals
  • Engineered Bioproducts

8
1. Basic Chemicals Organic Acids Citric
Acid 2-hydroxy-1,2,3-propane-tricarboxylic or
beta-hydroxytricarballylic acid. As part of the
tricarboxylic acid (TCA) cycle Citrate synthase
catalyses the reaction between acetyl-CoA and
oxaloacatate to form citric acid
9
The TCA cycle
10
(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.
11
(Citric Acid) It is a solid at room
temperature, Melts at 153C, Taste of various
fruits in which it occurs, e.g., lemons, limes,
oranges, Citric acid loses water at 175 C to
form aconitic acid, HOOCCHC(COOH) (CH2COOH),
which loses carbon dioxide to yield citraconic
anhydride
12
(Citric Acid) Itaconic anhydride rearranges to
citraconic anhydride (see Fig) or adds water to
form itaconic acid , (HOOCCH2 ) (HOOC)CCH2 Add
water to Citraconic anhydride gives citraconic
acid, cis-HOOCCHC(CH3) (COOH). Evaporation of
a citraconic acid solution in the presence of
nitric acid yields mesaconic acid, the trans
isomer of citraconic acid.
13
H2O
H2O
Itaconic Acid
Citraconic Acid
14
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
15
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.
16
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.
17
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.
18
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.
19
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.
20
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.
21
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
22
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.
23
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.
24
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.
25
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.
26
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.
27
Citric Acid Submerged Fermentation (6)
The fermenter is sterilised, charged with
substrate, and inoculated. Fermentation requires
3 14 days. After it is completed, the air
supply is stopped to prevent the microorganisms
from consuming the citric acid.
28
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.
29
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
30
Dissolved Citric Acid Lime ? calcium citrate
(ppt) ? filtered and stirred in dilute sulfuric
acid ? calcium sulfate (ppt) ? filtration
? purified citric acid solution
31
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

32
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
33
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, 427429.
  • 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.

34
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.
35
(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.
36
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).
37
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).
38
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.
39
  • 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)

40
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42
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.
43
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.
44
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 46
days to complete, and lactate yields of
approximately 90 wt from a dextrose equivalent
of carbohydrate are obtained.
45
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.
46
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
value-added applications.
47
Lactic acid Fermentation (5)
48
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49
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,
    90102 (1965).
  • Biomass Process Handbook, Technical Insights,
    Inc., Fort Lee, NJ., 1982, 96103.

50
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.
51
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.
52
1,3 Propanediol (3) 1,3-Propanediol readily
forms ethers. 3,3-Dihydroxydipropyl ether forms
upon continued reflux of the diol. 1,3-Propanedio
l reacts with aldehydes and ketones, often in the
presence of acidic catalysts, to form
1,3-dioxanes
53
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)
54
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  particularl
y 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 nickel-supported catalysts
at 2  4 MPa and 110 150 C in the organic
phase. The diol is subsequently separated from
solvent and water by distillation.
55
1,3 Propanediol (6) The yield of desired product
by this route is approximately 45 . Hydroformyla
tion 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.
56
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
57
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).

58
Amino Acids
  • 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

59
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
  • 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

60
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)

61
Glutamic Acid An amino acid, HOOCCH2CH2CH(NH2)CO
OH, 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 compound toxic
to the body.
62
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.
63
  • 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.

64
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.
65
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
66
Production
67
Extraction of Amino Acid
68
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.
69
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.
70
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.
71
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
ultra-filtration Absorbing and then collecting
lysine in an ion exchange step Crystallising or
spray drying of lysine as l-lysine hydrochloride
72
Lysine
  • References
  • http//www.adhd-becalmd.com/neurotransmitters/8/l-
    lysine-amino-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.

73
Major Classes of Bioproducts (Products derived
from bio-sources or used in bio-applications)
  • Basic Chemicals
  • Biochemicals
  • Biopharmaceuticals
  • Engineered Bioproducts

74
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.
75
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)
76
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.
77
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 13) 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.
78
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
79
Proteolytic Enzymes (cont). 5.
Chymotrypsin Found in pancreatic juice Catalyses
the hydrolysis of proteins into poly-peptides 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.
80
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.
81
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. Et
hyl 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)
  • Basic Chemicals
  • Biochemicals
  • Biopharmaceuticals
  • 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 Hav
ing 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
  • Synthetic Peptides
  • 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. Doesnt 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 are lacking
an enzyme (hypoxanthine guanine phosphoribosyl
transferase (HGPRT)). 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 recommended for children
at risk for the virus.
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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. Butthere is the risk of
allergic (Immune) reaction Rapid
inactivation Cant 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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