CEN 551 Biochemical Engineering

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CEN 551Biochemical Engineering

• Instructor Dr. Christine Kelly

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(No Transcript)
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Growing the microorganisms

• Medium substance used to grow the
  microorganisms. (Media is the plural.)
• In Class Assignment
• Form groups (not the same as usual).
• Write on a piece of paper what components should
  be in a typical microbial growth medium, and
  relative amounts (lots, medium, small, tiny)?

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Rich and Minimal Media

• Rich (complex) media yeast extract, corn steep
  liquor, Luria, Bacto. Complex mixtures of
  carbon, nitrogen, viatmins, amino acids, trace
  minerals. Generally unknown concentrations.
• Minimal (defined) media glucose, ammonium
  sulfate, magnesium chloride, potassium
  hyperchlorate, others in known proportions (often
  have to provide vitamins and minerals metals).

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Macronutrients

• Carbon
• Nitrogen
• Phosphorous
• Oxygen
• Hydrogen
• Sulfur
• Magnesium
• Potassium

• When in doubt
• CNP
• 100101

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Micronutrients

• Mo2
• Zn2
• Cu2
• Mn2
• Ca2
• Na2
• Vitamins
• Growth hormones
• Metabolic precursers

• Often a concentrated stock solution of vitamins
  and/or metals is used in growth medium.
• metal ions in media may require a chelating
  agent to form soluable compounds (ETDA a common
  chelating agent)

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Classification Scheme

• Heterotrophs organic compounds as carbon and
  energy source
• Autotrophs carbon dioxide as carbon source

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Industrial Sources of Carbon

• starch waste (potato, corn)
• molasses (cane and beet)
• corn sugar, corn steep liquor
• whey
• can use pure sugars (glucose, fructose) if
  product is valuable

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Nitrogen

• Some organisms fix nitrogen from N2 in the
  atmosphere
• Most use ammonium salts, proteins, amino acids

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Industrial Sources of Nitrogen

• Corn-steep liquor
• Yeast extract (expensive)
• Fish meal
• Dried blood

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Biotechnology Products
Enzymes are named by adding ase to the end of
the substrate (reactant) or reaction. For
example, urease and alcohol dehydrogenase.
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Review
Small molecules, proteins, and a ribosome drawn
to scale. Ribosomes are part of the machinery the
cell uses to make proteins each ribosome is
composed of about 90 macromolecules (protein and
RNA molecules).
http//gened.emc.maricopa.edu/bio/bio181/BIOBK/Bio
BookEnzym.html
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http//gened.emc.maricopa.edu/bio/bio181/BIOBK/Bio
BookEnzym.html
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Enzyme Function

• In order to do its work, an enzyme must unite -
  even if ever so briefly - with at least one of
  the reactants. In most cases, the forces that
  hold the enzyme and its substrate are
  noncovalent, an assortment of
• hydrogen bonds
• ionic interactions
• and hydrophobic interactions

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Active Site
http//gened.emc.maricopa.edu/bio/bio181/BIOBK/Bio
BookEnzym.html
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Active Site
http//gened.emc.maricopa.edu/bio/bio181/BIOBK/Bio
BookEnzym.html
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Specificity

• The requirement for complementarity in the
  configuration of substrate and enzyme explains
  the remarkable specificity of most enzymes.
  Generally, a given enzyme is able to catalyze
  only a single chemical reaction or, at most, a
  few reactions involving substrates sharing the
  same general structure.

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How Enzymes Work
http//gened.emc.maricopa.edu/bio/bio181/BIOBK/Bio
BookEnzym.html
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Co-Factors

• Many enzymes require the presence of an
  additional, nonprotein, cofactor.
• Some of these are metal ions such as Zn2 (the
  cofactor for carbonic anhydrase), Cu2, Mn2, K,
  and Na.
• Some cofactors are small organic molecules called
  coenzymes. The B vitamins
• thiamine (B1)
• riboflavin (B2) and
• nicotinamide
• are precursors of coenzymes.

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• Coenzymes may be covalently bound to the protein
  part (called the apoenzyme) of enzymes as a
  prosthetic group. Others bind more loosely and,
  in fact, may bind only transiently to the enzyme
  as it performs its catalytic act.

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Naming of Enzymes

• The International Union of Biochemistry (I.U.B.)
  initiated standards of enzyme nomenclature which
  recommend that enzyme names indicate both the
  substrate acted upon and the type of reaction
  catalyzed. Under this system, the enzyme uricase
  is called urate O2 oxidoreductase, while the
  enzyme glutamic oxaloacetic transaminase (GOT) is
  called L-aspartate 2-oxoglutarate
  aminotransferase.

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• Enzymes can be classified by the kind of chemical
  reaction catalyzed.
• Addition or removal of water
• Hydrolases - these include esterases,
  carbohydrases, nucleases, deaminases, amidases,
  and proteases
• Hydrases such as fumarase, enolase, aconitase and
  carbonic anhydrase
• http//www.worthington-biochem.com/introBiochem/li
  feProcesses.htmlnaming

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• Transfer of electrons
• Oxidases
• Dehydrogenases
• Transfer of a radical
• Transglycosidases - of monosaccharides
• Transphosphorylases and phosphomutases - of a
  phosphate group
• Transaminases - of amino group
• Transmethylases - of a methyl group
• Transacetylases - of an acetyl group

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• Splitting or forming a C-C bond
• Desmolases
• Changing geometry or structure of a molecule
• Isomerases
• Joining two molecules through hydrolysis of
  pyrophosphate bond in ATP or other tri-phosphate
• Ligases

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Enzyme Production

• Typically produced in cell lines designed for
  overexpression under fermentation conditions that
  favor expression.
• Enzymes can be expressed extracellularly (outside
  the cell), intracellularly (inside the cell), or
  both.
• Recovery of proteins expressed intracellularly
  requires cell lysis.
• Primary cost is separation and purification.

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Process Steps in Protein Recovery

• Cell lysis
• Gross debris removal
• Protein precipitation
• Salt removal
• Chromatographic separation
• Drying

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Cell Lysis

• membrane permeablization (CaCl)
• sonication
• French press
• grinding/bead beating
• freeze-fracture
• osmotic pressure

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Gross Debris Removal

• Microfiltration (hollow fiber filter, tangential
  flow filter)
• Centrifugation
• Protein precipitation
• Salting-out with inorganic salts (ammonium
  sulfate)
• Addition of organic solvents (MeOH) at low
  temperature

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Salt Removal

• dialysis
• size exclusion chromatography
• reverse osmosis
• electrodialysis
• ultrafiltration with molecular weight cut-off
  filters

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Chromatographic separation

• Adsorption
• Ion-exchange chromatography
• Gel filtration
• Affinity chromatography
• Crystallization
• Drying

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Uses of Enzymes

• Food Production
• Cheese (rennet)
• Beverage brewing (amylase, trypsin)
• Meat tenderizers
• Tofu production
• Wine-making (pectinase)
• Lactose removal in milk (lactase)

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Uses of Enzymes

• Chemical production
• detergents
• Fructose production from glucose (glucose
  isomerase)
• Amino acid production
• Vitamin production
• Optical resolution of DL amino acid mixtures
• Glucose production from starch
• Aspartic acid
• Urocanic acid production (UV block)
• Ethanol production

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Medical Uses

• Antibiotic production
• Blood purification
• Treatment of metabolic disorders
• Wound healing and antibacterial agents
• Treatment of stomach problems
• Blood clot removal
• Anticancer medicines

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Chemical Analysis and Process Monitoring

• Enzymatic chemical assays
• Immobilized enzyme electrodes

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Most Enzymes are Proteins

• The ribosomes are catalytic. They catalyze the
  formation of peptide bonds between amino acids.
  Ribosomes are composed of RNA and protein.
  However, the active part of the ribosome is RNA.
  Rare example on non-protein enzymes.
• http//users.rcn.com/jkimball.ma.ultranet/BiologyPa
  ges/E/Enzymes.html

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Examples of enzymes

• Catalase
• catalyzes the decomposition of hydrogen peroxide
  into water and oxygen. 2H2O2 -gt 2H2O O2
• One molecule of catalase can break 40 million
  molecules of hydrogen peroxide each second.

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• Carbonic anhydrase.
• Found in red blood cells where it catalyzes the
  reaction CO2 H2O lt-gt H2CO3
• It enables red blood cells to transport carbon
  dioxide from the tissues to the lungs.
  Discussion
• One molecule of carbonic anhydrase can process
  one million molecules of CO2 each second.

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• Acetylcholinesterase.
• Catalyzes the breakdown of the neurotransmitter
  acetylcholine at several types of synapses as
  well as at the neuromuscular junction - the
  specialized synapse that triggers the contraction
  of skeletal muscle.
• One molecule of acetylcholinesterase breaks down
  25,000 molecules of acetylcholine each second.
  This speed makes possible the rapid "resetting"
  of the synapse for transmission of another nerve
  impulse.

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Enzyme Kinetics

• Enzymatic reaction
• E S ES E P

Rate expression for product formation v dP/dt
k2(ES)
d(ES)/dt k1(E)(S)-k-1(ES)-k2(ES)
Conservation of enzyme (E) (E0) (ES)
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Two Methods to Proceed

• Rapid equilibrium assumption define equilibrium
  coefficient
• Km k-1/k1 ES/ES
• Quasi-steady state assumption
• ES k1ES/(k-1k2)
• Both methods yield the same final equation

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Michaelis- Menten Kinetics
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Michaelis-Menten Kinetics

• When v 1/2 Vmax, S Km so Km is sometimes
  called the half-saturation constant and sometimes
  the Michaelis constant

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Michaelis-Menten Kinetics

• units on k2 are amount product per amount of
  enzyme per unit time (also called the turnover
  number). Units on E0 are amount of enzyme
  (moles, grams, units, etc.) per unit volume
• Km has the same units as S (mole/liter, etc.)

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Experimentally Determining Rate Parameters for
Michaelis-Menten Kinetics Lineweaver-Burk
Eadie-Hofstee Hanes- WoolfBatch Kinetics
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In Class Assignment

• Form groups of 3-4
• Write on a piece of paper.
• 35 words or phrases that you think I expect you
  to be able to define without looking at notes or
  the text. Try to choose the words or phrases I
  would select.

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• Micronutrients
• CNP ratio
• Heterotrophs
• Autotrophs
• Enzyme
• Active site
• Activation energy
• Cell lysis
• Steps in protein purification
• Uses of enzymes

• Plasmid
• Procaryote
• Eucaryote
• Virus
• Ribosome
• Cytoplasm
• Central dogma
• Replication
• Transcription
• Translation
• Rich versus minimal media
• macronutrients

• Protein
• Lipid
• Amino acid
• Primary
• Secondary
• Tertiary
• Antibody
• Membrane
• DNA
• RNA
• Nucleotide
• base

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Project

• We will work together to define your project for
  this course. Begin by selecting a pharmaceutical
  product or therapy or disease that you are
  interested in. If you have selected a disease,
  we can then identify biologically produced
  products or therapies that target that disease.
  Some examples of diseases are Heart disease,
  Tuberculoses, Aids, Diabetes, Leukemia, other
  Cancers, Multiple sclerosis, and Alzheimers.
  After we have identified the drug or therapy, we
  will narrow the focus of your design. The design
  project may consist of a reactor system, a
  separation system, etc.

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• About one half of the project report will discuss
  the disease, the drug or therapy, the current or
  proposed manufacturing process, the current state
  of the drug or therapy (clinical trials,
  commercial product, prospective candidate), the
  current manufacturer, the patent activity, and
  the actual or estimated sales. The other half
  will be you design of some aspect of the
  manufacturing process. Included in the design
  will be your specifications for amount of product
  you are producing and why you selected that rate,
  type of equipment, size of equipment, detailed
  description of equipment, associated
  instrumentation, process control strategy, brief
  plan for validation of the equipment, cleaning
  and sterilizing procedures, typical operating
  problems you might expect and operating
  procedures.

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• Please select a project topic as soon as possible
  (within 2 weeks) and we will begin to work
  together to define your project. Send me you
  selections and ideas by email (ckelly_at_syr.edu).

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Possible Project Topics

• Amevive, a treatment for psoriasis from Biogen
  Inc.
• Avonex, Biogen Inc.
• Millennium Pharmaceuticals Inc. won approval of
  Velcade, a treatment for types of cancer in 2003.
  
• Cubist Pharmaceuticals Inc. of Lexington received
  approval for Cubicin, an antibiotic for stubborn
  infections in 2003.

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• Genzyme Inc. of Cambridge won two FDA approvals
  in 2003. Fabrazyme for treatment of Fabry
  disease, and
• Genzyme and partner BioMarin won approval for
  Aldurazyme to treat MPS 1, a deadly condition
  caused by an enzyme deficiency.
• Taxol, insulin, antibiotics