This action might not be possible to undo. Are you sure you want to continue?
T CENG 361 INTRODUCTION TO BIOCHEMICAL ENGINEERING AND BIOPROCESSSING
BIOCHEMICAL ENGINEERING PRODUCTS
LECTURE SLIDES PRESENTED TO BIEN STUDENTS PREPARED BY C.K. YEUNG
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).
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.
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 .
Lysine) 2. Esters) 5 . Lactic Acid) Alcohols (1.3 Propanediol) Amino Acids (Glutamic Acid. Biochemicals Enzymes (Proteolytic Enzymes) Surfactants (Lecithin. Basic Chemicals Organic Acids (Citric Acid.1.
Engineered Products Bio-devices (Bio-devices. Biopharmaceuticals Antibiotics (Penicillin) Monoclonal/Polyclonal Antibodies Hormones (Growth Hormones) Vaccines (Hep B Vaccine) Therapeutic Proteins (tPA) 4. Microorganisms DNA microarray chips.3. tissue/cell based) 6 .
Major Classes of Bioproducts (Products derived from bio-sources or used in bio-applications) 1)Basic Chemicals 1)Biochemicals 2)Biopharmaceuticals 3)Engineered Bioproducts 7 .
1. Basic Chemicals Organic Acids Citric Acid 2-hydroxy-1.3-propane-tricarboxylic or betahydroxytricarballylic acid.2. 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 .
anhydrous. an organic carboxylic acid containing three carboxyl groups.(Citric Acid) HO2CCH2C(OH)(CO2H)CH2CO2H. 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.
Taste of various fruits in which it occurs. limes. which loses carbon dioxide to yield citraconic anhydride 11 . e.. oranges. HOOCCH=C(COOH) (CH2COOH). lemons.(Citric Acid) It is a solid at room temperature. Melts at 153°C.g. Citric acid loses water at 175 °C to form aconitic acid.
(HOOCCH2 ) (HOOC)C=CH2 Add water to Citraconic anhydride: gives citraconic acid. cis-HOOCCH=C(CH3) (COOH).(Citric Acid) Itaconic anhydride rearranges to citraconic anhydride (see Fig) or adds water to form itaconic acid . the trans isomer of citraconic acid. Evaporation of a citraconic acid solution in the presence of nitric acid yields mesaconic acid. 12 .
+H2O +H2O Itaconic Acid Citraconic Acid 13 .
Citric acid can be recovered from its calcium salt by adding sulfuric acid.Citric Acid – Source and Production Can be extracted from the juice of citrus fruits by adding calcium oxide (lime) to form calcium citrate. It is obtained also by fermentation of glucose with the aid of the mold Aspergillus 14 . Precipitate can be collected by filtration.
15 . which employs a strain of Aspergillus niger to convert sugar to citric acid.The most economical method for producing citric acid since the 1930s has been fermentation. Both surface fermentation and submerged fermentation have been used.
sterile air at a controlled temperature.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. 16 .
or cane sugars. The substrate for the fermentation is a carbohydrate. refined beet. 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. or a syrup. usually a sugar. such as raw beet.
potato. (3) magnesium sulfate. such as (1) ammonium nitrate. (2) potassium phosphate. (4) zinc sulfate. 18 . The sugar content of the syrup can vary from about 10 to 25 wt %. corn. or other starch. and (5) potassium ferrocyanide. are added.Citric Acid – Surface Fermentation (3) Glucose syrups can be prepared from wheat. Certain inorganic nutrients.
Sterilisation may be batchwise or continuous. depending on the carbohydrate source. 19 . the latter uses less energy and is usually faster. After sterilisation.Citric Acid – Surface Fermentation (4) The pH is adjusted to between 3 and 7. the temperature is adjusted as required.
continuing at almost a constant rate until 80 – 90 % of the sugar is consumed. 20 . After two to three days the surface is completely covered and citric acid production begins. Fermentation then continues more slowly for an additional six to ten days.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.
niger uses some sugar for growth and respiration. (2) Particular strain of organism. However.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. the A. depending on such factors as: (1) Substrate purity. and the actual yield varies between 57 and 77 % of theoretical value. and (3) Control of fermentation 21 .
but takes place in large fermentation tanks.Citric Acid – Submerged Fermentation (1) Submerged fermentation is similar to surface fermentation. 22 . Equipment costs are also lower. This method is used more frequently because labour costs are lower with large tanks than with small pans.
a re-circulation loop. or a nozzle through which air and re-circulated substrate is pumped. and equipped with mixing devices. 23 . such as top-entering or side-entering agitators of the turbine or propeller type. Agitation can be increased by use of a draft tube.Citric Acid – Submerged Fermentation (2) The fermentation vessel can be short and wide or tall and narrow.
Oxygen is usually recovered from the exhaust gas. 24 . the air is cooled. which may be enriched with oxygen.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. if necessary. The air is supplied by a compressor and passes through a sterile filter.
the vessel must be equipped with heat-exchange surfaces. which can be the outside walls or internal coils. 25 . inoculum. sampling and exhaust ports are also provided. Ports are provided for introducing substrate.Citric Acid – Submerged Fermentation (4) Because the process is exothermic. and steam or other sterilising agents.
more commonly.Citric Acid – Submerged Fermentation (5) The substrate is prepared in a separate tank and its pH adjusted. by a continuous operation. 26 . The substrate is sterilised by a batchwise or. The micronutrients may be added to this tank or directly to the fermenter.
After it is completed. the air supply is stopped to prevent the microorganisms from consuming the citric acid. and inoculated.Citric Acid – Submerged Fermentation (6) The fermenter is sterilised. 27 . charged with Fermentation requires 3 –14 days. substrate.
or by centrifugation. 28 . First. The solids are washed to improve recovery of citric acid. biological solids usually are removed by filtration using a rotary vacuum filter or the more recent belt-press filter.Citric Acid – Recovery (1) The citric acid broth from the surface or submerged fermentation processes must be purified.
Citric Acid – Recovery (2) The dissolved citric acid must then be separated from residual sugars. and other soluble impurities. which is filtered and stirred in dilute sulfuric acid to form a precipitate of calcium sulfate. proteins generated by the fermentation. Addition of lime precipitates calcium citrate. filtration yields a purified citric acid solution 29 . This has traditionally been accomplished by precipitation and crystallisation.
Dissolved Citric Acid + Lime ↓ calcium citrate (ppt) ↓ filtered and stirred in dilute sulfuric acid ↓ calcium sulfate (ppt) ↓ filtration ↓ purified citric acid solution 30 .
The mother liquors are recycled to remove accumulated impurities 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.
is citric acid 32 .g.Citric Acid – Its use Can be obtained synthetically from acetone or glycerol. the citrates. e. Citric acid is used in soft drinks (45%) and in laxatives and cathartics. used in cooking. have many uses. ferric ammonium citrate is used in making blueprint paper. Its salts. Sour salt..
CRC Handbook of Chemistry and Physics. 11th ed. 1989. 1972.. – G. 456 – 465. Shreve's Chemical Process Industries. Weast. p. C. vol. Van Nostrand Co.. Fla. Dellweg (ed. Weinheim 1983. 163. 69th ed. N. Seidell... pp. Kominek: “Citric Acid” in H. and Primary Products. New York. Solubilities of Inorganic and Organic Compounds. CRC Press. Austin.. 33 . Verlag Chemie. 5th ed.. 1988 CRC Handbook of Chemistry and Physics.J. Merck & Co... DE-OS 2 240 723. Biomass.Citric Acid • Reference – The Merck Index. Boca Raton. – Ethyl Corp. 3. P.. J. C. Vol. 1989. Rahway. 2. Inc. – M. – A. Rohr. D.. McGraw-Hill Book Co.): Biotechnology Microbiology Products. 1941. Kubicek. 1984. 427– 429. 3rd ed.. New York. T. Inc. – R.
CH3CHOHCO2H. is the most widely occurring hydroxycarboxylic acid A colorless liquid organic acid. Miscible with water or ethanol. 34 .Lactic acid It was first discovered in 1780 by the Swedish chemist Scheele.
from anaerobic prokaryotes to humans Anhydrous lactic acid is a white. it is available as a dilute or concentrated aqueous solution. Lactic acid is also a principal metabolic intermediate in most living organisms.(Lactic acid) Lactic acid is a naturally occurring organic acid that can be produced by fermentation or chemical synthesis. crystalline solid with a low melting point. 35 . Generally.
yogurt. a soluble lactic acid salt. Calcium lactate. 36 .Lactic acid – Its use (1) A fermentation product of lactose (milk sugar) Is produced in muscles during intense activity. and cottage cheese (in situ microbial fermentation). is used as a source of calcium in the die Present in sour milk.
Lactic acid – Its use (2) The protein in milk is coagulated (curdled = go bad!) by lactic acid. inks. 37 . in leather tanning and textile dyeing. and in making plastics. Lactic acid is produced commercially for use in pharmaceuticals and foods. and lacquers (paint / natural varnishes a solution of cellulose derivative). solvents.
Lactic acid – Production (1) Although it can be prepared by chemical synthesis. 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. production of lactic acid by fermentation of glucose and other substances is a less expensive method. a dextro and a levo form. Chemically. lactic acid occurs as two optical isomers. .
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) 39 .• Lactic acid is the simplest hydroxy acid that is optically active.
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 (2) The commercial process is based on lactonitrile which used to be a by-product of acrylonitrile synthesis.
This crude lactic acid is esterified with methanol. producing the corresponding ammonium salt as a by-product.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 methyl lactate (see next slide) 41 .
purified.Lactic acid – Production (4) The latter is recovered and purified by distillation and hydrolysed by water under acid catalysts to produce lactic acid. and methanol. which is recycled. which is further concentrated. and shipped under different product classifications. 42 .
etc. leichmanii. whey. L. dextrose. corn syrup. Other complex nutrients required by the organisms are provided by corn steep liquor. molasses. A wide variety of carbohydrate sources.Lactic acid – Fermentation (1) The existing commercial production processes use homolactic organisms such as Lactobacillus delbrueckii. yeast extract. eg. and L. soy hydrolysate. bulgaricus. can be used. 43 . and cane or beet sugar.
and lactate yields of approximately 90 wt% from a dextrose equivalent of carbohydrate are obtained. 44 . taking 4–6 days to complete.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.
45 . 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%.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.
and food-grade lactic acid. and other valueadded applications. evaporated.Lactic acid – Fermentation (4) The calcium lactate-containing broth is filtered to remove cells. but not a heat-stable product. which is removed by filtration (See Figure) The filtrate is further purified by carbon columns and ion exchange and evaporated to produce technical. carbon-treated. which is required for the stearoyl lactylates. 46 . polymers. and acidified with sulfuric acid to convert the salt into lactic acid and insoluble calcium sulfate.
Lactic acid – Fermentation (5) 47 .
. C. New York. Inc. 3rd ed.. C. 90–102 (1965). – R. NJ. Chem.. Inc. 87. A. Lactic Acid. Prescott and C. – S. G. Holten. Rehbinder.. H. 1971. Muller. – Biomass Process Handbook. Technical Insights. Copenhagen. Fort Lee.. 1982. Schulz and J.Lactic Acid • References – C. International Research Association. and D. Industrial Microbiology. Schwaab. Denmark. 1959. McGraw-Hill Book Co. 49 . Verlag Chemie. 96–103. Dunn. Makromol.
is a clear. colorless.3 Propanediol (PDO) – Properties 1. and formamide. The chemical properties of 1. HOCH2CH2CH2OH. odorless liquid that is miscible with water.3-Propanediol. ethers. 50 .Alcohols 1. alcohols.3-propanediol are typical of alcohols. trimethylene glycol. It is sparingly soluble in benzene and chloroform.
Unlike 1.1.2-propanediol. respectively. 1. 51 .3-propanediol has two primary hydroxyl groups with equivalent reactivity.3 Propanediol (2) It reacts with isocyanates and acid chlorides to yield urethanes and esters.
often in the presence of acidic catalysts.3-dioxanes: 52 . 1.3 Propanediol (3) 1.1.3-Propanediol reacts with aldehydes and ketones.3Dihydroxydipropyl ether forms upon continued reflux of the diol.3-Propanediol readily forms ethers. 3. to form 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.
however. 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.1. 54 . the preferred technique is to extract the aldehyde into an organic solvent — particularly 2-methylpropanol — and then hydrogenate the aldehyde to yield the diol.3 Propanediol (5) 3-Hydroxypropanal can be hydrogenated in the aqueous phase directly.
3-propanediol in good yield (92 %). Hydroformylation of ethylene oxide followed by hydrogenation yields 1.3 Propanediol (6) The yield of desired product by this route is approximately 45 %.1. but a high catalyst concentration and a very large excess of solvent render the process uneconomical. 55 .
The reaction of ethylene with formaldehyde and carboxylic acids has also not been commericialised because of low selectivity 56 .1.3-propanediol with a rhodium – phosphine catalyst system has been disclosed.3 Propanediol (7) More recently. hydroformylation of ethylene oxide directly to 1.
G. p. 1970 (C. K. J. O. 93. L. D. – Hoechst Celanese. An.). Mateo.. Koetitz. 1980 (D. Schrauzer. 178 – L. 1989 (M. 2. R. Sastre. Abstr.1. F. 9. New York 1967. R. 19. Ser C 80 (1984) no. N. Soedin. 4th ed. US 3 463 910. 1981.. Murphy et al. Horvitz. Chem. US 4 322 355. – R. 425 – 432 – J. W. Interscience. N. 95 (1981) 20 039 e. – National Distillers and Chemical Corp. Lenz: Organic Chemistry of Synthetic High Polymers. Quim. 1182. 57 . Smith. W. no.3-Propanediol • Reference – Ullmann. Khim Geterotsikl. Lapuka et al. – Shell Oil Company. F. RuizMurillo.. US 4 873 378. Windgassen). Bargh).
which is an amino group (basic) an –H hydrogen a residue R which varies depending on the amino acid 58 .Amino Acids • • 1) 2) 1) 2) Amino acids are the building blocks of proteins There is –COOH. which is a carboxyl group (acidic) -NH2.
shape. charge. which is a carboxyl group (acidic) -NH2. hydrophobicity and reactivity –COOH. which is an amino group (basic) a –H hydrogen a residue R which varies depending on the amino acid 1) 2) 1) 2) 59 .Amino Acids (cont) • All 20 essential amino acids have this same structure but their side chain groups ‘R’ may vary in size.
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 .
a 61 compound toxic to the body. 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. HOOCCH2CH2CH(NH2)COOH.Glutamic Acid: An amino acid. .
Only the l-stereoisomer appears in mammalian protein.Lysine: Organic compound. 62 . one of the 20 AAs commonly found in animal proteins. Young adults need about 23 mg of this amino acid per day per kilogram (10 mg per lb) of body weight. The human body cannot synthesise it from simpler metabolites.
poultry.2. Sources of lysine include meats. and sometimes appears in the active site.6-Diaminohexanoic acid C6H14N2O2 • Has a net positive charge at physiological pH values making it one of the three basic amino acids. fish. • This polar amino acid is commonly found on the surfaces of proteins and enzymes. and dairy products. 63 .
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. 64 . wheat gluten. for example. is relatively poor in lysine.Lysine is found in particularly low concentrations in the proteins of cereals. .kwashiorkor Attempts to develop lysine-rich corn have been partly successful.
It also plays an important role in the functioning of histones. thereby aiding its solubility in water. its basic side chain often provides a positive electrical charge to the protein. and biotin) to enzymes. lipoic acid.Once lysine is incorporated into protein. Its side chain has also been implicated in the binding of several coenzymes (pyridoxal phosphate. The amino acid was first isolated from casein (milk protein) in 1889 65 .
Production 66 .
Extraction of Amino Acid 67 .
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. a gram-positive bacterium.Production by Fermentation (1) The most potent microorganisms to overproduce lysine are mutants derived from Corynebacterium glutamicum. 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 .
Fermentation processes are performed in big tanks up to 500 m3 size.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. 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 .
W. 72 .-H. 50 (1986) 339 – 346. – Y. O.). Tosaka. US 4 623 623.adhd-becalmd. H. Bioprocess. Yoshi. Liu. Karasawa. Tsao.-T.com/neurotransmitters/8/l-lysine-amino-acid – M. Biol. Wu.Lysine • References – http://www. Chem. Eng. 1986 (T. Nakanashi et al. J. S. 9 (1993) 135 – 139. Ikeda. Agric. – Kyowa Hakko.-C.
Major Classes of Bioproducts (Products derived from bio-sources or used in bio-applications) 1)Basic Chemicals 1)Biochemicals 1)Biopharmaceuticals 2)Engineered Bioproducts 73 .
Enzymes are made from amino acids. 74 . and they are proteins. Biochemicals Enzymes The purpose of an enzyme in a cell is to allow the cell to carry out chemical reactions very quickly.an enzyme acts as a very efficient catalyst for a specific chemical reaction.2. When an enzyme is formed. The chain of amino acids then folds into a unique shape. it is made by stringing together between 100 and 1.000 amino acids in a very specific and unique order. That shape allows the enzyme to carry out specific chemical reactions -.
Trypsin (enzyme of the pancreatic juice.Proteolytic Enzymes 1. Renin (secreted by the kidneys that is involved in the release of angiotensin) 2. capable of converting proteins into peptone) 75 .
or proteolytic. enzymes in the GIT. these enzymes. Pepsin Enzyme produced in the mucosal lining of the stomach that acts to degrade protein. Pepsin is one of three principal protein-degrading..Proteolytic Enzymes (cont).e. During the process of digestion. i. peptides and AAs. 3. break down dietary proteins to their components. 76 . the other two being chymotrypsin and trypsin.
3.Proteolytic Enzymes (cont). also produced by the gastric mucosa. Pepsin and other proteolytic enzymes are used in the laboratory analysis of various proteins. 77 . hydrochloric acid. is necessary to convert the inactive enzyme and to maintain the optimum acidity (pH 1–3) for pepsin function. pepsin is also used in the preparation of cheese and other protein-containing foods. Pepsin (cont) Pepsin is synthesised in an inactive form by the stomach lining.
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).
Subtilisin Produced by the bacterium Bacillus subtilis. used as an active ingredient in detergents and also in research to help reveal protein structure. 5.Proteolytic Enzymes (cont). 79 . Chymotrypsin Found in pancreatic juice Catalyses the hydrolysis of proteins into polypeptides and amino acids 6.
Cathepsin Intracellular proteolytic enzymes. 7. kidneys. esp. and intestine. spleen. that catalyze autolysis in certain pathological conditions and after death. the liver. occurring in animal tissue. Fibrinolysin (also known as plasmin) Formed in the blood from plasminogen. that causes the breakdown of the fibrin in blood clots 8. 80 .Proteolytic Enzymes (cont).
81 . occurring in animal and plant tissues and egg yolk. and egg yolk. phosphoric acid. composed of units of choline. corn. Lecithin Group of phospholipids. used in foods. A commercial form of this substance. fatty acids. and glycerol. obtained chiefly from soybeans.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. cosmetics. and inks.
82 .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. wetting. and antioxidant properties 2. have emulsifying. 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.
2. When ethanol and acetic acid react. 83 . ethyl acetate (an ester) and water are formed. extracts. formed by the reaction between methanol and acetic acid. and lacquers. Ethyl acetate is used as a solvent. Esters (cont). is a sweet-smelling liquid used in making perfumes. Methyl acetate. the reaction is called esterification.
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.
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.
Major Classes of Bioproducts (Products derived from bio-sources or used in bio-applications)
1)Basic Chemicals 2)Biochemicals 1)Biopharmaceuticals 1)Engineered Bioproducts
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 87 .
Penicillin G and Penicillin V 88 . floxacillin. Includes: Cloxacillin.Antibiotics Penicillins (cont)…. ampicillin.
cats and dogs. 2 types are commonly used: 1) Synthetic Peptides 2) Fusion Proteins 89 . goats etc) with a suitable immunogen.Antibodies Methods of Obtaining Antibodies • Traditionally. antibodies to human gene products have traditionally been obtained by repeatedly injecting suitable animals (rodents. rabbits.
90 .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.
each of different specificity 91 .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.
it will undergo conformational change. The idea is that when conjugated to a suitable molecule. Doesn’t really work! (It is difficult to generate suitably specific antibodies) 93 .Synthetic Peptides The amino acid sequence is inspected and a synthetic peptide (often 20-50 amino acids long) is designed. This will then adopt a structure resembling the native polypeptide.
94 .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.
antibodies should be secreted into the serum specific The antibody-rich serum (antiserum) which is collected contains a heterogeneous mixture of antibodies.Antibodies (Polyclonal) If the animal system has responded. each produced by a different B lymphocyte The different antibodies recognise different parts (Epitopes) of the immunogen (Polyclonal Antisera) 95 .
96 . or hybridoma (see next series of slides for details). multiplies rapidly. Monoclonal antibodies are typically made by fusing a normally short-lived.Antibodies (Monoclonal) An antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. such as a cancer cell. creating a clone that produces large quantities of the antibody. The resulting hybrid cell. antibody-producing B cell to a fast-growing cell.
each can provide a permanent and stable source of a single type of monoclonal antibody (mAb) (See Figure included) 97 .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.
Hybridoma (cont). 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. 99 . Animals produce highly heterogeneous mixtures of antibodies (Ab 1. the hybrid clones can be grown in unlimited quantity in vitro or can be grown as tumours in recipient animals.
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.Hybridoma (cont). cultures are grown in the so-called HAT (Hypoxanthine. Upon fusion. Thymidine) selective medium. Aminopterin. The myeolma cells (hypoxanthine guanine (HGPRT)). 100 .
allowing the hybrid to grow in HAT medium. 101 . The immunocytes from hyperimmune animals provide the genetic material for the production of a specific antibody and HGPRT. Non-fused parental myeloma will disappear in HAT while non-fused immunocytes are over grown by the hybrids.Hybridoma (cont).
Transgenic mice have been engineered to contain human immunoglobulin loci permitting the in vivo production of fully human antibodies. 102 .Genetically-Engineered Antibodies DNA cutting and ligation technology could be used to generate new antibodies including both partially humanised antibodies and fully humanised antibodies.
103 . adrenals. where it exerts some influence upon the metabolism of the target tissue. Produced and secreted by the endocrine glands including the pituitary. testes. pancreatic islets. thyroid.Hormones Secretary substance carried from one gland or organ of the body via the bloodstream to more or less specific tissues. ovaries. among the mammalian species. parathyroids. certain portions of the gastrointestinal tract. and the placenta.
and oxytocin. The thyroids secrete thyroxine and calcitonin. and the parathyroids secrete parathyroid hormone. the gonadotropic hormones and growth hormone The posterior pituitary secretes antidiuretic hormone.Hormones The anterior pituitary include thyrotropin. prolactin. adrenocorticotropic hormone. 104 .
Hormones The adrenal medulla secretes epinephrine and norepinephrine while the cortex of the same gland releases aldosterone. and cortisone. and testes in fact produce at least small amounts of all of the steroid hormones. 105 . cortisol. The ovaries primarily secrete estrogen and progesterone and the testes testosterone. The adrenal cortex. corticosterone. ovaries.
and somatostatin. which produces erythrocytes (red blood cells) 106 . glucagon.Hormones The islets of Langerhans in the pancreas secrete insulin. The kidneys also produce erythropoietin.
Human Growth Hormones Growth hormone or somatotropin . 107 . Evidence suggests that the secretion of human growth hormone (HGH) is regulated by the release of certain peptides by the hypothalamus of the brain. glycoprotein hormone released by the anterior pituitary gland that is necessary for normal skeletal growth in humans.
108 . but its most obvious effect is the stimulation of the growth of cartilage and bone in children.Human Growth Hormones (cont). HGH is known to act upon many aspects of cellular metabolism. has been shown to inhibit the secretion of HGH. called somatostatin. One such substance.
as killed or weakened bacteria or viruses. to stimulate antibody production 109 .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.
A vaccine was made available in 1995 and is 110 recommended for children at risk for the virus. . Spreads by physical contact The disease usually resolves on its own. the virus being present in feces and transmittable via contaminated food or water. Exposed persons can be protected by injections of gamma globulin.Vaccines (Hepatitis) Hep A Infectious hepatitis. occurs sporadically or in epidemics.
Some infected individuals. particularly children. Intravenous-drug abusers remain a high-risk group Spread by sexual transmission and from mother to baby at birth. was commonly transmitted through blood transfusions. become chronic carriers of the virus.Hep B Serum hepatitis. 111 .
HepB can progress to chronic liver disease and is associated with an increased risk of developing liver cancer. is recommended for all infants and others at risk for the virus.Hep B (Cont). Alpha-interferon was approved as a treatment in 1992 112 . A vaccine. available since 1981.
non-B hepatitis Is also transmitted by contaminated blood transfusions and by sharing of needles. Many of those infected have no symptoms but become carriers 113 .Hep C Formerly called non-A. It is the most common form of chronic liver disease in the US.
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.
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.
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)
Coagulation factors VIII (Haemophilia A). γInterferon (chronic granulomatous disease) etc. growth factors and some can act as biocatalysts or inhibitors Huge impact on physiological processes Examples: α-Interferon (HepB Vaccine). DNase (cystic fibrosis) etc… 117 .Therapeutic Proteins (cont) Gene technology aided the production of large amount of protein drugs tPAs such as hormones.