Biosepartaion Engineering: Ch.1: Bioseparation & Biological Materials

KAIST Bioseparation Eng.
Biosepartaion engineering
Ch.1: Bioseparation & biological materials
Hyun Gyu Park
Ch. 1: Bioseparation & biological materials

KAIST 1.1 Introduction Bioseparation Eng.
Bioseparation: separation and purification of bioproducts (compounds of biological origin)

 Consist of a sequence of recovery and separation steps that maximize the purity of the
bioproduct while minimizing the process time, yield losses, and costs
Bioproducts: chemical substances or combination of chemical substances that are made by living
things from methanol to whole cells
 Must be extracted and purified to some degree before being suitable for market
 Choice of separation method: the nature of the product

KAIST
Unit operations Bioseparation Eng.
 Removal of solids
• Filtration, Centrifugation, Sedimentation
 Recovery or Isolation (volume reduction)

• Adsorption, Extraction
 Purification
• Chromatography. electrophoresis, precipitation
• Chromatography  over half of the purification costs of biopharmaceuticals
 Polishing
• Crystallization, Drying

 World production levels and prices of bioproducts (Fig. 1.1)

 Erithropoietin (EPO)  Amgen & Genentech $800,000/g

 Erythropoietin or EPO is a glycoprotein hormone that controls red blood cell production. It is a cytokine for
erythrocyte (red blood cell) precursors in the bone marrow. It is produced by the kidney in response to low oxygen
pressure in the blood. (Amgen & Genentech $800,000/g)
 Interferons (IFNs) are natural cell-signaling proteins produced by the cells of the immune system of most
vertebrates in response to challenges such as viruses, parasites and tumor cells. Interferons assist the immune
response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing
antigen presentation to lymphocytes, and inducing the resistance of host cells to viral infection. The protein
interferon, produced by animal cells when they are invaded by viruses, is released into the bloodstream or
intercellular fluid to induce healthy cells to manufacture an enzyme that counters the infection. Interferon is
therefore considered a potential medical resource as a BIOPHARMACEUTICAL. For many years the supply of
human interferon for research was limited by costly extraction techniques. In 1980, however, the protein became
available in greater quantities through GENETIC ENGINEERING. -interferon has been approved for therapeutic
use against hairy-cell LEUKEMIA and Hepatitis C.
 Vinblastine is an anti-mitotic drug used to treat certain kinds of cancer, including Hodgkin's lymphoma, non-
small cell lung cancer, breast cancer, head and neck cancer, and testicular cancer. Vinblastin: Vinblastin/vincristin
from Catharanthus roses acts as cell-cycle inhibitor and thus can be used as anticancer drugs.
 Digitalis is also known as digoxin and digitoxin. It's a drug that strengthens the contraction of the heart
muscle, slows the heart rate and helps eliminate fluid from body tissues. It's often used to treat congestive heart
failure and is also used to treat certain arrhythmias. Digitalis has been described in medical literature for over 200
years. It's derived from the foxglove plant.

 Use in Molecular Biology: Digoxigenin
 Digoxigenin (DIG) is a steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea and
Digitalis lanata. It is used as a molecular probe to detect DNA or RNA. It can easily be attached to nucleotides by
chemical modifications. DIG molecules are often linked to uridine nucleotides; DIG labeled uridine (DIG-U) can
then be incorporated into RNA probes via in vitro transcription. Once hybridisation occurs in situ, RNA probes
with the incorporated DIG-U can be detected with anti-DIG antibodies that are conjugated to alkaline phosphatase.
To reveal the hybridised transcripts, alkaline phosphatase can be reacted with a chromogen to produce a colour
precipitate.
 Pectinase is a general term for enzymes that break down pectin, a polysaccharide substrate that is found in the
cell walls of plants. One of the most studied and widely used commercial pectinases is polygalacturonase. It is
useful because pectin is the jelly-like matrix which helps cement plant cells together and in which other cell wall
components, such as cellulose fibrils, are embedded. Therefore pectinase enzymes are commonly used in processes
involving the degradation of plant materials, such as speeding up the extraction of fruit juice from fruit, including
apples and sapota. Pectinases have also been used in wine production since the 1960s. They can be extracted from
fungi such as Aspergillus niger. The fungus produces these enzymes to break down the middle lamella in plants so
that it can extract nutrients from the plant tissues and insert fungal hyphae. If pectinase is boiled it is denatured
(distorted) making it harder to connect with the pectin at the active site, and produce as much juice.

KAIST General Bioprocess Bioseparation Eng.
Feedstock Bioprocessing Product
GAS Cell culture

PRODUCT
Recovery
LIQUID Biocatalyst Bioreactor
product LINES
SOLID Enzymatic
Feedstock Bioprocessing Product

 Gas  Immobilized Enzymes  Separation  Pharmaceuticals
− Syn. Gas − Ambient to Extreme − In situ  Fine chemicals
− CO2  Fermentation − Secondary  Specialty Chemicals
− Organic vapor − Immobilized  Media  Feedstock
 Liquid − Free cell − Gaseous  Bulk chemicals
− Organic − Ambient to Extreme − Aqueous
− Sugar solution  Bioreactors − Organic
 Solid − Continuous Systems
− Biomass − Membrane
− Consumer Waste − Batch or Fed-batch

KAIST 1.2 Broad classification of bioproducts Bioseparation Eng.
 Small molecules: amino acids, sugar, fine chemicals, antibiotics, hormones, vitamins
 can not be sedimented, but can be separated by extraction
 Lipids
 Large molecules: proteins, polysaccharides, nucleic acids

 highly adsorptive
 Particulate products
 can be collected by sedimentation or filtration

KAIST Length Scale Bioseparation Eng.
1 cm
Insects
1 mm
Hair
100 m Red Blood Cells
Bacteria
10 m
1 m
100 nm
Virus
Ribosome
10 nm Antibodies
Amino Acids
1 nm
Sucrose
Water
0.1 nm Carbon-carbon bond

Hydrogen bond

KAIST Biomolecules of Interest Bioseparation Eng.
4.5 nm
2.4 nm 14 nm
DNA Lysozyme ATP synthase

KAIST 1.3 Small molecules Bioseparation Eng.
Naturally occurring compounds and metabolites such as sugar, citric acid, vitamins, amino
acids, lipid, and antibiotics
1.3.1 Primary Metabolites

 Formed during the primary growth phase of the organism
 Key metabolic Intermediates are used in catabolism & anabolism

 Key metabolic Intermediates are used in catabolism & anabolism
 Catabolism: the process by which microorganisms obtain energy from organic compounds.
Catabolism is the metabolic process that breaks down molecules into smaller units. It is made up
of degradative chemical reactions in the living cell. Large polymeric molecules (polysaccharides,
nucleic acids and proteins) are processed into their constituent monomeric units (i.e.
monosaccharides, nucleotides and amino acids, respectively).
 Anabolism (biosynthesis) is the metabolic process that builds larger molecules from smaller
ones. Anabolic processes tend toward "building up" organs and tissues. These processes produce
growth and differentiation of cells and increase in body size, a process that involves synthesis of
complex molecules. Examples of anabolic processes include growth and mineralization of bone
and increase of muscle mass.


 Sugar: monosaccharides, disaccharides, polysaccharides
sucrose from sugar cane in food industry (world’s highest tonnage bioproduct)
glucose: common fermentation nutrient
Sugars as products in bioprocess
 mannitol to fructose (partial oxidation by acetic acid bacteria)
 glucose to fructose (glucose isomerase)
 fructose corn syrup: sweeteners in soft drinks
CH2OH CH2OH
O OH H O H CH2OH H
H O
H H
H OH H H HO
OH
HO H HO O CH2OH
OH H
H OH H OH
Glucose Fructose
Sucrose


 Organic alcohols, acids, and ketone
 can be produced by the anaerobic fermentation of microorganisms
 ethanol, isopropanol, acetone, acetic acid, lactic acid, propionic acid
 Vinegar is the acetic acid product produced by Acetobacter bacteria with ethanol as a
substrate
 Vitamins: “vital amine”

 Vitamin C, ascorbic acid
 Human & guinea pig do not synthesize vitamin C while nearly all other mammals do
 Required to support the hydroxylation of proline in collagen
 Support antioxidant functions and sulfation of cholesterol
 Natural antioxidants: tocopherol (vitamin E) & glutathione
 Excellent example of a present-day market tension: natural vs synthetic
 Most vitamins can be synthesized in organic chemical reaction (harmful impurities)
 Extraction from plants and fermentation produces vitamins naturally – at higher cost
Ascorbic acid, Vitamin C Biotin, Vitamin H


 Amino acids
 Building blocks of proteins
 Zwitterionic at neutral pH:
 carry positive & negative charges simultaneously
 List of amino acids: structure, abbreviations, pK of R group (Table 1.3)
* pH= -log10a H+ & k (ionization constant)=[A+][B-]/[AB] (AB  A+ + B- )
 All the -amino acids contain asymmetric carbon atom
 except for glycine (plane-polarized light)
 Levo rotatory(-) from living things, Dextro rotatory(+)
 Dehydration condensation  Peptide bond formation by ribosome

Hydrophobic Amino Acids

 amino acids (0.5 nm)
Hydrophilic Amino Acids
Special Amino Acids

 Amino acids  Specific properties of amino acid side group

 Acidic or basic AAs: adsorption by ion exchange or separation by electrophoresis
 Many aliphatic side chains: preferential adsorption onto or extraction into nonpolar media
 Free –SH (sulfhydryl) can be used to bind proteins to immobilized mercury
 Histidine forms coordination complexes with metals (Ni++)


 Lipids
 Lipids can be broadly defined as any fat-soluble (hydrophobic) naturally-occurring molecules, that
are insoluble in water but soluble in nonpolar organic solvents such as ether, benzene, chloroform.
 Highly extractable into nonpolar solvents
 Although the term lipid is sometimes used as a synonym for fat, it is in fact a subgroup of lipids
called triglycerides.
 The term is more-specifically used to refer to fatty-acids and their derivatives (including tri-, di-, and
monoglycerides and phospholipids) as well as other fat-soluble sterol-containing metabolites such as
cholesterol.
 Fatty acid are usually esterified to glycerol to form di- & triglycerides
 Diglycerides are usually esterified to a phosphate group (phospholipids), which may in turn be
esterified to ethanolamine or choline
 Fatty acids and phospholipids are amphiphilic with a strongly polar “head” & a strongly nonpolar
“tail”


 Lipids
 Lipid (Greek: lipos, fat): organic molecule of biological origin that is insoluble in water and soluble
in nonpolar solvents
 Hydrophobic molecules & smaller than other polymeric molecules
 Found mostly in fatty tissues, membranes, and other nonpolar biological structures
 Structural components of membranes
 Cell-surface components concerned in cell recognition and tissue immunity
 Storage and transport forms of metabolic fuel (fats)
 Form hybrid molecules such as glycolipids and lipoproteins
 Some lipids have intense biological activity (vitamins & hormones)
 General Categories of Lipids

 Fatty acids
 Triacylglycerols
 Phospholipids
 Steroids
 Waxes
 Lipophilic vitamins
Lipids: fatty acids Bioseparation Eng.
KAIST
Fatty acid: unbranched carboxylic acid

 12-20 carbons most common
 Most biologically-important fatty acids have 18 carbons: stearic, oleic, and linoleic acids
 Main biological function: component of other lipids
 Categorized by C=C in chain: saturated (no C=C) or unsaturated (one or more C=C)
Some important unsaturated fatty acids

O O
OH
OH
Linoleic acid (C18)

Oleic acid (C18)
O O
OH
OH
Linolenic acid (C18)

Arachidonic acid (C20)

Lipids: fatty acids Bioseparation Eng.
KAIST

Lipids: Triacylglycerols Bioseparation Eng.
KAIST
Triacylglycerol (triacylglyceride): fatty acid triester of glycerol (glycerin)
 Most abundant natural lipids
 Triacylglycerol = fat if solid at room temperature; oil if liquid
 Main biological function: energy storage
 Hydrolysis (“water breaking”) of animal fats yields soap
NaOH, H2O
heat
OH + RCO2- Na+
OH + R'CO2- Na+
OH + R"CO2- Na+
 Saturated fat ☞ packed side by side (solid at RT) mixture = soap

 Unsaturated fat ☞ cause bending in carbon skeleton

Lipids: Triacylglycerols Bioseparation Eng.
KAIST
How does soap work?

 Hydrophilic CO2- groups attracted to d+ H-O-H d+
 Nonpolar (hydrophobic) hydrocarbon chains avoid water
 Nonpolar (lipophilic) hydrocarbon chains attracted to nonpolar “dirt”, other fatty acid chains
 Forms micelles (~spherical aggregates); suspends “dirt” in water
 Micelles carry dirt away when wash water is removed
-
O O
O O
-
-
Free soap Hydrophobic tail O O
molecules in H2O dirt

O
O O
-
Hydrophilic CO2-
-
O O
O
-
-
- O O
O - O O O - O
O O O O
-
-
-
O
O O Micelle
dirt
Hydrophobic tails dissolve in nonpolar “dirt”

Lipids: Phospholipids Bioseparation Eng.
KAIST
 Glycerol esterified with two fatty acids and one phosphate group
 Second most abundant group of natural lipids
 Fatty acids and phospholipids are amphiphilic with a strongly polar “head” & a strongly nonpolar “tail”

Lipids: Phospholipids Bioseparation Eng.
KAIST
Main biological function: cell membranes (phospholipid bilayer)

Lipids: Steroids Bioseparation Eng.
KAIST
 Steroid: a molecule having the ring system shown below

 Composed of 4 fused carbon rings
 Shape: fairly flat and fairly rigid
 Steroids have similar structures but wide range of functions
 Functions
 part of membranes
 hormones
 development
 ion balance
H3C
C D H3C H
A B H H
HO
Steroid example: cholesterol

Steroid skeleton
Cholesterol is biological precursor to all other steroids

Lipids: Steroids Bioseparation Eng.
KAIST

Lipids: Lipophilic Vitamins Bioseparation Eng.
KAIST
Vitamin: an organic compound, other than fat, protein or carbohydrate, required for the normal
growth and maintenance of animals
 Very broad range of structures and functions
Vitamin A (retinol)
• Essential to vision OH
• Incorporated into rhodopsin (photon-harvesting protein)
Vitamin E
• Mixture of isomers; α-tocopherol most important
• Protects against oxidative damage to cells from radicals
CH3
HO
CH3 CH3
CH3 O
CH3
CH3
a-Tocopherol
Hydrophobic antioxidant vitamin

 Commercial uses of primary metabolites (Table 1.4)
 Low molecular weight compounds: easy to purify owing to their thermal stability
 Some of the alcohols like ethanol: purification using distillation

1.3.2 Secondary Metabolites

 Produced at or near the beginning of the stationary phage
 Antibiotics: Organic substances produced by microorganisms which have the ability to destroy or
inhibit the growth of bacteria and other microorganisms
 Penicillin from Penicillium notatum, 1928 Fleming
 Penicillin binds at the active site of the transpeptidase enzyme that cross-links the peptidoglycan
strands. The labile β-lactam ring in penicillin reacts with a serine residue in the transpeptidase as
shown below. This reaction is irreversible and so the growth of the bacterial cell wall is inhibited.
The resulting complex is stable to water and remains attached to the polypeptide chain.

1.3.2 Secondary Metabolites

 LG Factive: Quinolone (FDA approval in April of 2003)
 The group of antibiotics known as the β-lactams include penicillins, cephalosporins, monobactams
and the carbapenems.
 their unique structural feature is the presence of the four-membered ß-lactam ring.

1.3.3 Summary of small biomolecules

 Methanol, acetone, aspirin: chemical synthesis has displaced extraction of natural products
 In many cases, bioprocess technology is economically superior
*Paclitaxel: anticancer compound

 made from the bark of the Pacific yew tree (Taxus brevifolia)
 approved by the U.S. FDA for treating breast and ovarian cancers as well as AIDS-related Kaposi's
sarcoma.
 Taxol: BMS (Bristol Myer Squibb) brand name
 Genexol: Samyang Genex
 It functions by immobilizing microtubules in dividing cells & preventing the redissociation of the
mitotic spindle
 Poor water solubility  hydrophilic or charged functionalities

KAIST 1.4 Macromolecules Bioseparation Eng.
 Proteins
 Polysaccharides
 Nucleic acids

Macromolecules
 Monomers are linked together to form polymers

 dehydration synthesis (condensation)
 broken apart via hydrolysis

Structure of Protein Bioseparation Eng.
KAIST
 Final 3-D structure of a single protein

 Folding pattern of H-bonds, hydrophobic
interactions, ionic bond, disulfide bridge
 Results from interactions between R groups
 Covalent amino acid sequence
 Regular, repeating patterns as a result of H-bonds

 α-Helix: H-bond between NH & CO (3 AA residues)
 β-Sheet: cross-linked by interchain H-bonds
 Formation of multimeric complexes by individual protein molecules

 Arrangement of protein subunits into a large macromolecule

Primary structure Bioseparation Eng.
KAIST
Amino acids All backbone bonds are covalent

There are no branched peptides despite the existence of
amino & COOH in side chains
Peptide bond

KAIST 1.4.2 Secondary structure (-helix) Bioseparation Eng.
- Formation of hydrogen bonds between oxygen and hydrogen atoms

-  helix: each NH to a CO group by H-bond at a distance equivalent to three AA residues
Alpha helix
 Coil held by hydrogen bonding every 4th AA
5.44Å
2.3Å
Bio.kaist.ac.kr/~hskim/ppt/nano_lecture.ppt & http://myhome.hanafos.com/~s9euno/ch3-1.htm

KAIST 1.4.2 Secondary structure (-Sheet) Bioseparation Eng.
  sheet: side-by-side polypeptide chains are cross linked by interchain hydrogen bonds
 Typical of fibrous proteins such as silk
 CD (circular dichroism): detect changes in the 2dary structure by measuring the difference in the abs.
of left circularly polarized light & right circularly polarized light  estimate the fraction of a protein
in  helix,  sheet, and random coil configurations
7.0
Å

Tertiary Structure
 Hydrophobic AAs fold inward owing to surrounding polar water molecules

 In globular proteins in water, many AAs with nonpolar side chains are forced to the inside
 The folding step: constrained by the strong covalent disulfide bonds between Cys
 Once formed, a tertiary structure is often stabilized by the formation of disulfide bridges

Tertiary Structure
Ser Val
Cys
Asp
Asp
Lys
Lys

KAIST 1.4.3 Tertiary structure Bioseparation Eng.
Specific overall shape of a protein

Cross links between R groups of amino acids in chain
 disulfide –S–S–
 ionic –COO– H3N+–
 H bonds C=O HO–
 hydrophobic –CH3 H3C–

KAIST Disulfide bond Bioseparation Eng.
Cysteine side chains are readily oxidized in air
2 R-S-H + O2  R-S-S-R + H2O2
2 R-S-H + H2O2  R-S-S-R + 2 H2O
4 R-S-H + O2  2 R-S-S-R + 2 H2O
Cleavage of Disulfide bridges
1) Performic acid oxidation

2) Sulfhydryl reducing agents
 Mercaptoethanol
 dithiothreitol or dithioerythritol (Cleland's reagent)

Breaking disulfide bonds in proteins Bioseparation Eng.
KAIST
OH
HS
SH
OH

KAIST 1.4.3 Tertiary structure Bioseparation Eng.
 Example 1.1 Effect of a reducing agent on protein structure and mobility

Upon the addition of 1mM dithiothreitol (a reducing agent)  decrease of a relative mobility of a protein
by 10% during the migration of the protein through a polyacrylamide gel. Why?
☞ DTT causes the disulfide bond in the protein to be broken. The breaking of the disulfide bonds
causes the protein to unfold and the 3D structure to become less compact  move more slowly
Reducing agent
 2-mercaptothanol: a mild reducing agent that is ideal for cleaving disulfide bonds to thiols.
 DTT: a water-soluble reagent that reduces disulfide bonds
OH
HS
SH
-mercaptoethanol OH
HSCH2CH(OH)CH(OH)CH2SH
Dithiothreitol

Quaternary Structure
 Folded peptide chains interact with one another in the native conformation of an oligomeric protein
 Hemoglobin molecule: 2 -globin and 2- globin peptide chains
 Bacterial RNA polymerase: several subunits
 Quaternary structure is maintained by intermolecular bonds: ionic & disulfide bond
Structure of Heme B
Heterocyclic organic ring, porphyrin
Prosthetic group

KAIST 1.4.5 Prosthetic groups and hybrid molecules Bioseparation Eng.
 The majority of proteins are conjugated proteins that contain not only AAs, but other organic and
inorganic compounds  Prosthetic group: non-amino acid portion of a conjugated protein
 Posttranslational modification: any changes to protein molecules other than the addition of AAs via
peptide bond formation
 Glycosylation: oligosaccharides are added to active side chains of specific AAs (Ser, Asn, Thr)
 Side chain oligosaccharide serve extremely important functions such as stability, catalytic function,
binding specificity, solubility, targeting, and transport
 Human EPO, which is normally made in cells of the kidney and transported via the blood to the bone
marrow  recombinant protein by animal or yeast cell culture (not by bacteriaglycosylation)
TABLE 1.5 Classification of Proteins According to Prosthetic Group

Class Prosthetic Group Example
β1-lipoprotein of blood
Lipoproteins Lipids
Glycoproteins Oligosaccharides Gamma globulin
Phosphoproteins Phosphate Protein kinase C
Hemoproteins Iron porphyrin Hemoglobin
Flavoproteins Flavin nucleotides Succinate dehydrogenase
Metalloproteins Iron, zinc Ferritin, alcohol dehydrogenase

Antibody
Antibody structure
 Globular proteins synthesized by B lymphocytes in animals
 Highest association constant with antigen
 Y-shaped protein molecules with an invariant stem
 Each pair of N-terminal residues constitutes an antigen binding site
 CH2O denotes carbohydrate
C: constant region
V: variable region
H: heavy chain
L: light chain
 Fc of heavy chain: [-chain: IgA] [-chain: IgD] [-chain: IgG] [-chain: IgM] [-chain: IgE]

Antibody
WBC: leukocytes
1. Lymphocyte
 B cell: synthesize antibody proteins
 T cell: Inflammatory T cell, cytotoxic T cell, helper T cell ( activate B cell, macrophage, etc.)
2. Monocyte: become macrophage
3. Granulocyte
 Epitope: a portion of a molecule to which an antibody binds. Epitopes can be composed of sugars,
lipids or amino acids. In most cases, epitope tags are constructed of amino acids. (antigenic
determinant)
Figure 9.2 Schematic representation of a target

antigen. The surface of the antigen depicted has
seven (numbered 1 to 7) different antigenic
determinants (epitopes). When this antigen is used to
immunize an animal, each antigenic determinant
elicits the synthesis of a different antibody. Together,
the different antibodies that interact with an antigen
constitute a polyclonal antibody directed against that
antigen.

KAIST 1.4.5 Prosthetic groups and hybrid molecules Bioseparation Eng.
Antibody: hybrid molecules that have become a major product of biotechnology
 Globular proteins synthesized by B lymphocytes in animals

 Genes that code for proteins are supposed to be constant and stable, but antibody genes are an exception:
they are susceptible to internal rearrangement of DNA base sequences that encode the “hypervariable”
region of antibody, the part that binds antigens
 Antigen generally has several different epitopes  produce a different antibody against one of the many
epitopes of the antigen  polyclonal antibody
 Polyclonal antibody: a number of different antibodies that would each bind to a different antigenic
determinant (epitope) on the target molecule
 Monoclonal antibody: a single type of antibody molecule from B lymphocytes that are members of a
“clone”
 How to create a cell line that could be grown in culture & would produce monoclonal antibody: a trick
has been devised for producing them in bioreactors
 B lymphocytes do not reproduce in culture
 Normal B lymphocytes sometimes become cancel cells (myelomas) that acquire the ability to grow in
culture while retaining many of the attributes of B cells
*Hybridoma hybridizing one B lymphocyte with a lymphoma (blood cell tumor) cell
Hybridoma, which grows in culture and secretes a single type of antibody molecules, is the source of a
monoclonal Ab.
Hybridomas produce progeny indefinitely in vitro which make monoclonal antibodies.

Immune system

Production of monoclonal antibodies
HW1 (Due date: Sep. 12)

Explain how to select and separate each
clone from the mixture of the clones.

Production of monoclonal antibodies

KAIST 1.4.6 Functions and commercial uses of proteins Bioseparation Eng.
Table 1.6 Classification of proteins according to function

Class Examples
Ribonuclease
Trypsin
Enzymes Urokinase
DNA polymerase
Cellulase
Hemoglobin
Serum albumin
Transport proteins
Myoglobin
β1-Lipoprotein
Ovalbumin
Nutrient, storage protein
Casein
Actin
Contractile or motile Myosin
Tubulin
Keratin (skin)
Fibroin (silk)
Structural Collagen (tendons, ligaments)
Elastin (joints)
Proteoglycans (cell walls)
Immunoglobulin (antibodies)
Venom proteins
Defense
Ricin
Interferon
Insulin
Growth hormone
Regulatory Lymphokines/cytokines
Protein kinases (signal transduction)
DNA binding repressors and activators
Soybean trypsin inhibitor
Inhibitors Plasminogen activator inhibitor
HIV protease inhibitor

KAIST 1.4.6 Functions and commercial uses of proteins Bioseparation Eng.
 Enzyme: catalysts of reactions

 Highly specific
 Active site binds substrate and contains the AA residues that are directly involved in the making
and breaking of bonds
 Protein purification: preservation of tertiary & quarternary structures
• Catalyst
E + S ↔ ES*→ E + P
• High specificity
 Regio-specificity
 Stereo-specificity
• High turnover rate
• Instability
 Heat
 Organic solvents
 Denaturants
Lysozyme-substrate complex
Lysozyme: The enzyme functions by attacking peptidoglycans (found in the cell walls of bacteria, especially Gram-positive
bacteria) and hydrolyzing the glycosidic bond.

KAIST 1.4.7 Stability of proteins Bioseparation Eng.
 Since the active function of proteins is dependent on tertiary and quaternary structures, the 3D
configuration of each molecule must be maintained
 Protein degradation
 deamidation of asparagine and glutamine
 oxidation of methionine to methionine sulfoxide form

 Methionine, cysteine, tryptophan, tyrosine and histidine residues are susceptible to oxidation. The oxidation
of methionine to the sulfoxide form is of particular interest as it has been shown to occur in a wide variety of
proteins and often reduces or eliminates biological activity.

 Since the active function of proteins is dependent on tertiary and quaternary structures, the 3D
configuration of each molecule must be maintained
 Protein degradation
 deamidation of asparagine and glutamine
 oxidation of methionine to methionine sulfoxide form
 oligomerization
 aggregation
 fragmentation (depolymerization)
 cross-linking
 inter- and intramolecular rearrangements involving cysteine disulfide exchanges
 denaturation: breakdown of H-bonds and ionic bonds that determine tertiary structure
 Analytical methods for QC

 Gel electrophoresis: changes in Mw  aggregation, oligomerization, fragmentation
 Isoelectric focusing: depends on charge  detect deamidation on the basis of the appearance of
dissociating amino groups
 HPLC: Information on both purity & structure
 Methods used to purify proteins are also used as analytical methods for QC

Variables that affect protein stability

☞ Temperature, pH, mechanical shear, chemical agents, irradiation
 Temperature
Thermal denaturation for many proteins begins to occur at 45 to 50 oC
An example of the thermal denaturation of the protein ribonuclease (Fig. 1.17)
 High temperature  break H-bond & -pleated sheet structure is disrupted
 Still leaves the disulfide bonds intact
Some enzymes from thermophilic bacteria inhabiting hot springs
 active at high temperature above 85oC

KAIST 1.4.8 Recombinant protein expression Bioseparation Eng.
 Two issues in the development of a purification process

for a recombinant protein
1. Should the protein be genetically engineered to aid in
the purification?
2. Must the protein be solubilized and refolded from
“inclusion bodies?
Heterologous proteins expressed in prokaryotic
cells often do not fold correctly leading to
precipitate called an “inclusion body”
Insoluble & inactive aggregates of protein inside
the bacterial cell when a protein is overproduced
in a recombinant bacterium.

Genetic engineering to improve protein purification

 Affinity peptide or protein tags  Fusion protein
 Histidine oligomer:
 six histidine tag binds transition metals such as nickel, zinc, copper
 His tag bind to metal ions immobilized in a purification medium such as chromatography column
 GSTglutathione
 MBPamylose: affinity tags
Target protein His tag
 Genetic engineering to avoid the expression of insoluble inclusion bodies  fusion protein
 Expressed as C-terminal fusion with another protein called a carrier protein (higher solubility)
 Carrier proteins: GST (glutathione-S-transferase), MBP (maltose binding protein), NusA protein,
thioredoxin

Use of Fusion protein : Affinity tag
Buffer flow
Target protein His tag Ni
Non-His tag
fused proteins
Resin : Ni-matrix
 Ni ion interact with Histidine hexamer
 His-tag fused protein retains in the column (Ni-resin)
 The other non-his tag fused proteins are washed away
 Finally, His-tag fused target proteins are eluted from the column purely

Use of Fusion protein : Affinity tag
Buffer flow
Target protein His tag Ni
Elution of His-tag
fused proteins
Resin : Ni-matrix
Ni ion interact with Histidine hexamer
His-tag fused protein retains in the column (Ni-resin)
the other non-his tag fused proteins are washed away
Finally, His-tag fused target proteins are eluted from the column purely
☞ Imidazole (the side chain of histidine)

Use of Fusion proteins
Figure 6.11 Immunoaffinity chromatographic

purification of a fusion protein. An antibody
that binds to the marker peptide of the fusion
protein (anti-marker peptide antibody) is
attached to a solid polypropylene support.
The secreted proteins are passed through the
column containing the bound antibody. The
marker peptide portion of the fusion protein
is bound to the antibody, and the other
proteins pass through. The immunopurified
fusion protein can then be selectively eluted
from the column by the addition of pure
marker peptide.
 Target protein removal from fusion

 by proteases such as bovine enterokinase
 by self-cleaving proteins called inteins

Protein folding Bioseparation Eng.
KAIST
 Many of the proteins produced in E.coli  Insoluble, intracellular, biologically inactive inclusion body
 In vivo insolubility of protein  incorrect folding
 Expensive & time-consuming protein solubilizing and refolding procedures
What are inclusion bodies ?
Insoluble, inactive, protease resistant, dense clumps of aggregated,

misfolded protein
When do they form ?
When (foreign) protein is overexpressed (aggregation of refolding

intermediates)
Inclusion body purification
Solubilisation (6M GnHCl, 8M urea) and Renaturation

KAIST

KAIST

Bacterial culture sample Containing IB

(inclusion body)
Centrifuge 6000rpm, 10 min, 4℃
Supernatant Cell pellet Resuspend pellet in PBS
Sonication 3s/5s (on/off), 2min,

20% amplitude
Supernatant Cell debris & Viable cells
0.5~1% Triton X-100

10000rpm, 10 min, 4℃
containing buffer
Soluble fraction Insoluble Detergent
Centrifuge IB pellet
fraction washing
Solubilization and refolding of proteins in inclusion bodies

The agglomerated protein must be resolubilized by strong denaturants
The protein must be refolded to its correct 3-D conformation
Solubilization
After cell lysis & centrifugation, the proteins in inclusion bodies are usually solubilized by adding
chaotropic agent such as guanidinium chloride or urea
A Chaotropic agent is an agent which causes molecular structure to be disrupted; in particular,

those formed by nonbonding forces such as hydrogen bonding, Van der Waals interactions, and the
hydrophobic effect.
The most commonly used chaotropes are 6~8 M urea and 6 M guanidinium chloride, with urea
being an uncharged molecule and guanidinium chloride being a hydrochloride salt.
For protein with cysteine, add a reducing agent such as -mercaptoethanol, dithiothreitol (DTT),
dithioerythritol, or cysteine to allow reduction of the interchain disulfide bonds by thiol-disulfide
exchange
NH2C(:NH)NH2·HCl
NH2C(O)NH2
Guanidine hydrochloride
Urea

Solubilization and refolding of proteins in inclusion bodies

Refolding
Performed by dialysis or dilution
Dialysis: use of ultrafiltration membrane in a large
volume of renaturation buffer
Dialysis can be scaled up by means of diafiltration 
adding renaturation buffer to the denatured protein
solution to keep the volume constant
 During refolding to the native state N, kinetic

competition of folding & aggregation of denatured state
D ☞ N ⇔D → aggregates
 To minimize aggregation
☞ refolding usually must be performed at low conc.
(<100mg/l) (Fig. 1.20)
For proteins with disulfide bonds
 renaturation buffer with a redox system
 add reduced or oxidized forms of low Mw thiol
reagents (glutathione, cysteine, cysteamine)
 allow the formation and reshuffling of disulfides


1.5 Macromolecules: Nucleic Acids Bioseparation Eng.
KAIST
 Nucleic Acids store and transmit hereditary information

 DNA ☞ stores the information in a ‘genetic code’
 RNA ☞ carries the information to the protein synthesizing
machinery
 Nucleic acids are chemically more robust than proteins
 Their 2ndary structures are highly stable, being based on base-
pairing hydrogen bond
 Nucleic acids are hydrolyzed by enzymes that are ubiquitous
in nature
 RNA in particular is susceptible to ribonuclease molecules that
seem to be everywhere
 Width of double-helical strand : 2.0 nm

 Distance between adjacent base pairs : 0.34 nm
 Number of bases per one round : 10 bases → 3.4 nm

KAIST Structure of DNA Bioseparation Eng.
 Structure of DNA
 James Watson & Francis Crick at 1953
 Native DNA consists of two long chains (strands) that form a
double-stranded helix
 Coiled polynucleotide chains of DNA: hydrogen bonds between
the bases
 Base pairing rule: A-T(U) & G-C
 A-T: two hydrogen bonds & G-C: three hydrogen bonds
- Wallace rule (less than 15mer): Tm = 2oC (A+T) + 4oC (G+C)
 Kilobase pairs (kb) or megabase pairs (Mb)

 Antiparallel orientation  the two strands of a duplex DNA run
in opposite directions to each other

KAIST Structure of DNA Bioseparation Eng.

Nucleic Acids Bioseparation Eng.
KAIST
Nucleotide: nitrogenous base, ribose (deoxyribose) sugar, one or more phosphate groups

KAIST

KAIST

KAIST
RNA can be:

Single stranded
Double stranded
Hybridized with DNA

KAIST
The DNA double helix and its replication
AUG: Start codon ☞ methionine

UAA, UAG, UGA: Stop codon
1.6 Macromolecules: Polysaccharides Bioseparation Eng.
KAIST
Carbohydrates
 Sugars and polymers of sugar
 Have carbonyl and multiple hydroxyl groups
 Primarily C, H, and O  1:2:1 ratio of C:H:O ☞ (CH2O)n
 Appears to be “carbon hydrate” from formula
 4 categories: monosaccharides, disaccharides, oligosaccharides, polysaccharides
 Functions
 structure
 energy source
 cell to cell recognition
Polysaccharides
 Not necessarily linear like peptides & nucleic acids

 Numerous OH  possible branching via typical glycosidic linkage
 Most familiar polysaccharides: starch , glycogen, cellulose

KAIST Carbohydrates: polysaccharide Bioseparation Eng.
Starch & Glycogen

 Both are used for energy storage
 Starch = energy storage in plants
 Amylose(α-1,4, 20∼25%) & amylopectin(α-1,4 & α-1,6, 75∼80%)
 Potatoes & grains , wheat, rice, corn G
G G
GGG
 Glycogen = energy storage in animals G G
G
 α-1,4 & α-1,6 ☞ more branched & compact G
G
G G  1-6 link
 Found in humans and vertebrates G
G
 1-4 link G
 Stored in liver and muscles G
Glycogen
G
 Hydrolysis in the cells fulfill glucose requirements G
 Glycogen storages get depleted throughout the day

 When energy is needed, enzymes break the bonds between monosaccharides
 Branching allows for more points of access for enzymes to act (greater surface area)

Carbohydrates: polysaccharide
Cellulose & Chitin ☞ structural support

 Cellulose  structural support in plants
 Tough cell walls of plant cells
 Present in beta form whereas starch is in alpha form
 Chains are linked through hydrogen bonds of hydroxyl groups
 Humans don’t have any enzyme to digest cellulose
 Chitin  makes up the hard exoskeleton of insects
 Glucose monomer with attached nitrogen
 Arranged in beta configuration
 Leathery, flexible, and strong
 Used as surgical threads for suturing and decomposes easily once wound is
healed
 Long, unbranched chains
 Fibers form parallel chains
 Long, unbranched chains provide greater strength

Importance of Carbohydrates
 Distributed widely in nature
 Key intermediates of metabolism (sugars)
 Structural components of plants (cellulose)
 Central to materials of industrial products: paper, fibers
 Key component of food sources: sugars, flour, vegetable fiber
 Bacterial polysaccharides are the active component of a number of vaccines
 Microbial polysaccharide: xanthan gum, dextran, alginate, pullulan
 Cellulose, agarose, dextran are used in media for bioseparation

1.6 Macromolecules: Polysaccharides Bioseparation Eng.
KAIST
TABLE 1.8 Some Polysaccharides, Their Sources and Uses
Polysaccharide Name Polysaccharide Source Uses
Starch Potatoes. Corn Food, clothing
Cellulose Wood, cotton Paper, clothing
Carrageenam Mycophyta (algae) Food
Agar Mycophyta Microbiology, food
Dextran Corn Food, medicine
Agarose Mycophyta Biochemistry
Xanthan gum X. campestris Food, industrial chemical, oil field drilling

KAIST 1.7 Particulate Products Bioseparation Eng.
 Large molecules
• Proteins (3-10 nm)
• Polysaccharides (4-20 nm)
• Nucleic acids (2-1,000 nm)
 Particulate products
• Virus (100 nm)
• Bacteria (1um), yeast cells (4 um), animal cells (10 um)
• Liposomes or natural vesicles (100 nm)
• Bacterial inclusion bodies (100-1,000 nm)
• Subcellular particles or organelles  Ribosomes (25 nm)
• Natural hormone granules (200 nm)
• Generally purified by centrifuge

• Some very small particles  ultracentrifugation
 Subcellular components
• Early bioseparation steps include flocculation, sedimentation, filtration

KAIST 1.8 Introduction to Bioseparation: Engineering analysis Bioseparation Eng.
 Implementation of the biotechnological products or process: biochemical engineering

 upstream engineering: fermentation
 downstream engineering: purification or bioseparation
 unit operation such as sedimentation, adsorption, drying
 Important properties for bioseparation: thermal stability, solubilities, diffusivities, charge, isoelectric
pH, reaction rate constants, separation thermodynamics
1.8.1 Stages of downstream processing

 Objectives and Typical Unit Operations of the Four Stages in Bioseparations (TABLE 1.9)

1.8.2 Basic principles of engineering analysis

Governing equations: material balance, equilibria, flux (or transport phenomena)
 Material balance
accumulation = inflow - outflow + amount produced - amount consumed
 Equilibria
equilibrium constant for chemical reaction
Keq=[C]/([A][B]) for A + B  C
extraction process: partition coeff. K=y/x
y: conc. of a separand in the extract phase
x: conc. of the same separand in the raffinate (usually heavy) phase
adsorption process
CS: conc. in the adsorbent phase
C: conc. in the liquid phase (C: chemical species & S:adsorption site)
Keq=[CS]/[C]  linear adsorption equilibrium (valid at low conc.)

1.8.2 Basic principles of engineering analysis

Governing equations: material balance, equilibria, flux (or transport phenomena)
 Flux relationships (Transport phenomena)

- Flux = coefficient x driving force
flux: flowing per unit area per unit time
driving force: gradient down which units flow
coefficient: permeability or the inverse of a resistance (properties of medium)
- Ohm’s law: Je= CE
Je: current density
C: electrical conductivity
E: electrical potential gradient
- Fick’s first law for diffusive flux due to a concentration gradient dc/dx in one dimension
D: diffusion coefficient  property of the medium
In some cases, calculable from the Stokes-Einstein equation for spheres
JD = - D(dc/dx)
D = kT/6πμa (k: Boltzmann constant, T: absolute temp., μ: viscosity, a: particle radius)

1.8.3 Process and product quality

Measures of product quality
 Purity = amount of product/(amount of product + amount of total impurities)
 Product purity: different levels of purity are required for different products (Fig. 1.23)
 Specific activity = units of biological activity/mass
 Yield = amount of product produced/amount of product in feed
 Pyrogen: any substance that produce a fever (from gram negative bacteria, endotoxin)
 Endotoxin: toxins associated with certain bacteria. Classically, an "endotoxin" is a toxin that,
unlike an "exotoxin", is not secreted in soluble form by live bacteria, but is a structural
component in the bacteria which is released mainly when bacteria are lysed. The prototypical
examples of endotoxin are lipopolysaccharide (LPS) or lipo-oligo-saccharide (LOS) found in
the outer membrane of various Gram-negative bacteria and is an important cause of their ability
to cause disease.

Endotoxin: a pyrogenic (fever inducing) substance (e.g. lipopolysaccharide) present in the

bacterial cell wall. Endotoxin reactions range from fever to death.
http://pathmicro.med.sc.edu/fox/lps.jpg
Extremely heat stable – recommended conditions for inactivation are 180 0 C for 3 hours.

KAIST 1.9 The route to market Bioseparation Eng.
 New drug discovery: several years for the completion of the route (Fig. 1.24)
Preclinical trial: animal test, toxicity test
Phage I: safety for usually healthy adults
Phage II: efficacy for 20-80 patients
Phage III: efficacy for hundreds – thousands patients
Important factors for the route to market

1.9.1 The chemical and applications range of the bioproduct
Aspartame: dipeptide nonnutritive sweetener
Paclitaxel: triterpene from plants used in cancer treatment
Erythropoietin: peptide hormone that stimulates red blood cell production in the bone marrow
Oligonucleotide
Bacillus thuringiensis (Bt): whole bacterial cells that when dried can be sprayed on crops to prevent
insect damage
Food, medicine, agriproducts

Biosepartaion Engineering: Ch.1: Bioseparation & Biological Materials

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Biosepartaion Engineering: Ch.1: Bioseparation & Biological Materials

Încărcat de

Drepturi de autor:

Formate disponibile

KAIST Bioseparation Eng.

Ch.1: Bioseparation & biological materials

Hyun Gyu Park

Ch. 1: Bioseparation & biological materials

Bioseparation: separation and purification of bioproducts (compounds of biological origin)

Ch. 1: Bioseparation & biological materials

 Recovery or Isolation (volume reduction)

Ch. 1: Bioseparation & biological materials

 World production levels and prices of bioproducts (Fig. 1.1)

Ch. 1: Bioseparation & biological materials

Ch. 1: Bioseparation & biological materials

 Use in Molecular Biology: Digoxigenin

Ch. 1: Bioseparation & biological materials

Feedstock Bioprocessing Product

GAS Cell culture

Feedstock Bioprocessing Product

Ch. 1: Bioseparation & biological materials

 Large molecules: proteins, polysaccharides, nucleic acids

Ch. 1: Bioseparation & biological materials

100 m Red Blood Cells

0.1 nm Carbon-carbon bond

Ch. 1: Bioseparation & biological materials

DNA Lysozyme ATP synthase

Ch. 1: Bioseparation & biological materials

1.3.1 Primary Metabolites

Ch. 1: Bioseparation & biological materials

 Key metabolic Intermediates are used in catabolism & anabolism

Ch. 1: Bioseparation & biological materials

1.3.1 Primary Metabolites

Ch. 1: Bioseparation & biological materials

1.3.1 Primary Metabolites

 Vitamins: “vital amine”

Ascorbic acid, Vitamin C Biotin, Vitamin H

Ch. 1: Bioseparation & biological materials

1.3.1 Primary Metabolites

Ch. 1: Bioseparation & biological materials

Hydrophobic Amino Acids

Hydrophilic Amino Acids

Special Amino Acids

Ch. 1: Bioseparation & biological materials

1.3.1 Primary Metabolites

 Amino acids  Specific properties of amino acid side group

Ch. 1: Bioseparation & biological materials

1.3.1 Primary Metabolites

Ch. 1: Bioseparation & biological materials

1.3.1 Primary Metabolites

 General Categories of Lipids

Fatty acid: unbranched carboxylic acid

Some important unsaturated fatty acids

Linoleic acid (C18)

Linolenic acid (C18)

Ch. 1: Bioseparation & biological materials

Ch. 1: Bioseparation & biological materials

 Saturated fat ☞ packed side by side (solid at RT) mixture = soap

Ch. 1: Bioseparation & biological materials

How does soap work?

molecules in H2O dirt

Hydrophobic tails dissolve in nonpolar “dirt”

Ch. 1: Bioseparation & biological materials

Ch. 1: Bioseparation & biological materials

Main biological function: cell membranes (phospholipid bilayer)

Ch. 1: Bioseparation & biological materials

 Steroid: a molecule having the ring system shown below