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

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eferences: tkinson, B., and M. Ferda, Biochemical Engineering and Biotechnology Handbook, 2nd ed., Stockton Press, New York, NY (1991). ailey, J. E., and D. F. Ollis, Biochemical Engineering Fundamentals, 2nd ed., McGraw Hill, New York, NY (1986). lanch, H. W., and D. S. Clark, Biochemical Engineering, Marcel Dekker, New York, NY (1996). uerk, D. G., Biosensors: Theory and Applications, Technomic Publishing Company, Inc., Lancaster, PA (1993). ebelein, C. G., ed., Biotechnological Polymers, Technomic Publishing Company, Inc., Lancaster, PA (1993). all, E. A. H., Biosensors, Prentice Hall, Inc., Englewood Cliffs, NJ (1991).
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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.
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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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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)
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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)
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Major Classes of Bioproducts (Products derived from bio-sources or used in bio-applications)

1)Basic Chemicals 1)Biochemicals 2)Biopharmaceuticals 3)Engineered Bioproducts

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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
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The TCA cycle

(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.
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(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, HOOCCH=C(COOH) (CH2COOH), which loses carbon dioxide to yield citraconic anhydride
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(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.
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+H2O

+H2O

Itaconic Acid

Citraconic Acid
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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
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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.

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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.
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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.

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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.
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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.

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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.
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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
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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.
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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.

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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.
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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.

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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.

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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.
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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.
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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
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Dissolved Citric Acid + Lime calcium citrate (ppt) filtered and stirred in dilute sulfuric acid calcium sulfate (ppt) filtration purified citric acid solution
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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

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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
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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.
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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.
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(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.
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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).
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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).

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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 38 isomers.

 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 Dlactic acid (2)

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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)
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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.

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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 46 days to complete, and lactate yields of approximately 90 wt% from a dextrose equivalent of carbohydrate are obtained.
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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%.

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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.
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Lactic acid Fermentation (5)

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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.

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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.
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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.

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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:

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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)

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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.

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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

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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).
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Amino Acids
1) 2) 1) 2) 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
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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

1) 2) 1) 2)

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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)

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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.
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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.
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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.
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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
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Production

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Extraction of Amino Acid

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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.
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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.

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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.

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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-lysine-amino-acid 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.

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Major Classes of Bioproducts (Products derived from bio-sources or used in bio-applications)

1)Basic Chemicals 1)Biochemicals 1)Biopharmaceuticals 2)Engineered Bioproducts

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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)

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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.
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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.
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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

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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.
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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.
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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 1)Biopharmaceuticals 1)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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92

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, antibodies should be secreted into the serum specific

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. 96

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)

97

98

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. 99

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.
100

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 110 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. 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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