Microbes As Tools Flashcards
How Is Glucose Converted To Fructose? is The Process Efficient?
0.1M Glucose Treated with 1.22 M KOH at 5°C under Nitrogen for 3.5 Months
5% Yield Of Fructose
7% Unchanged
88% Glucose Converted To Hydroxy Acids
The Process Is Inefficient
Describe Conversion Of Glucose To Fructose In Eukaryotes.
First Stage Of Glycolysis
Requires: 3 Enzymes + ATP
Expensive
Describe Conversion Of Glucose To Fructose In Prokaryotes.
Requires 1 Enzyme
No Co-Factors
Cheap
Produces Almost 100% Yield
How Are Microbes Used In Industries?
Fermented Foods & Beverages
Alcoholic Drinks
Food Preservation
Enzyme Production (For Food Processing)
Dairy Products
Antibiotics
Alcohol fermentation: Anaerobic Process Microorganisms Convert Sugars → Ethanol + CO₂
- Glucose Converted Into (x2) Pyruvate In Cytoplasm of Yeast Cells
Net gain: 2 ATP + 2 NADH (Reducing agent)
- Pyruvate Decarboxylation
Pyruvate Decarboxylase Makes Pyruvate lose 1 Carbon As CO2, Leaving Acetaldehyde As Remaining Product
- Acetaldehyde Reduction
Alcohol Dehydrogenase Reduces Acetaldehyde → Ethanol via
NADH Donating Electrons To Acetaldehyde
NAD+ Regenerated → Used To Continue Glycolysis
Describe Acetic Acid Fermentation.
The Oxidation of Ethanol into Acetic Acid by Certain Aerobic bacteria
Requires: Oxygen
Mechanism:
- Alcohol Dehydrogenase Oxidizes Ethanol To Acetaldehyde
- Aldehyde Dehydrogenase Oxidizes Acetaldehyde To Acetic Acid
- Acetaldehyde Further Oxidized To Acetic Acid
How Are Lactic Acid Bacteria Used In Food Preservation?
- Acidification Of Environment
LAB Convert Sugars into Lactic Acid, Lowering the pH of Food
Low pH Inhibits Spoilage Bacteria + Pathogens
- Production Of Antimicrobial Compounds:
LAB Produce Bacteriocins → Proteins that Kill/Inhibit Other Bacteria
Food Preservation also carried out using Carbon Dioxide, Hydrogen peroxide Production
- Competitive Exclusions:
LAB Grow Quickly, Outcompete Harmful Microbes For Nutrient + Space
What Is Bioremediation?
Process That uses Microbes, Fungi, Plants/Enzymes To Degrade, Detoxify or remove environmental contaminants
Contaminants: Oil Spills, Heavy Metals, Pesticides, Industrial Waste
Describe Microbial Enzymes.
- Catalyze Reactions That Don’t Normally Occur
- Highly Specific + Selective,
- Normally Catalyze 1 Reaction
Can Be:
Regioselective – Discrimination Between Similar Parts Of Same Molecule
Stereoselective – Act on/ Generate single Optical Isomers
Conditions usually very mild
✓ less energy usage
✓ cheaper equipment
✓ safer
What Are Commercial Enzyme Sources?
- Animals
- Plants
- Bacteria
- Yeast
- Fungi
What Is Affinity Purification?
Biochemical Technique used To Isolate Specific Molecules From Complex Structures based On Highly Specific Binding Interaction
What Are Expression Hosts?
Living Cells In Which Foreign Genes Are Introduced, So The Cell Expresses The protein Encoded By The Gene
Why Are E. coli used as Expression Hosts?
Because They Have Simple, Well Characterized Genetics
They Are Inexpensive + Fast To Grow
There are Lots Of Vectors Available And Are Easily Manipulated
Evaluate The Use of E. coli as Expression Hosts
Cons:
- High Level Expression In Bacterial Cytosol
Overwhelms Folding Machinery - Causes Misfolding Of Protein/ Insoluble
Aggregates That Are Inactive
Pros:
- Expression Can Be Tuned/Regulated, By Co-
Expressing Chaperones + Lowering Induction
Temperature - Inclusion Bodies Easy To Pellet Out By
Centrifugation - Proteins Can Be solubilized + Refolded Into
Active Forms
Why Are Animal/Insect Cells Used As Expression Hosts?
Contain Extensive Chaperone Systems To Help Large/Multi-Domain Proteins Fold Correctly
ER Provides Oxidizing Environment 🡢 Allows Disulfide Bond Formation + Shuffling
Disulfide Bonds Needed For Antibodies, Receptors + Secreted Enzymes
Evaluate The use of Animal/Insect Cells As Expression Hosts.
Pros:
Secretory Pathways Allow Protein Collection Directly From culture Supernatant 🡢 Harsh Lysis Steps + Protease Damage Avoided
e.g. Signal-Peptide-Dependent ER 🡢Golgi 🡢 Secretory Vesicle
Provide Authentic Folding + Disulfide Bond Formation + Glycosylation Patterns 🡢 Needed For Therapeutic Proteins + Receptors
Cons:
Expensive Growth Medium
Low Yields (Inefficient)
🡣Robust 🡢 More Susceptible To Protein Overexpression
Susceptible To Contamination
Why Are Yeasts Used As Expression Hosts?
They Allow Functional Eukaryotic Protein Production As They Have ER + Golgi Based Machinery To form Disulfide Bonds + N-Linked Glycosylation
Allow Functional Eukaryotic Proteins Production Without Moving To Mammalian Cultures
Evaluate The Use Of Yeasts As Expression Hosts.
Pros:
They Have A Low Protein Production Cost
They Have A High Yield
Can be Easily Grown To High Densities
Cons:
Non-Human Glycosylation: Yeasts N-Glycans Differ From Those of Human Glycans 🡢 Impairs Protein Activity, half Life, Triggers Immune Responses In Therapeutic Proteins
Grow Slower Than Bacteria 🡢 Translates Into Lower Biomass + Volumetric Productivity
