Untitled Quiz

Enzyme Inhibition

• Enzyme inhibition is a process where a molecule (inhibitor) binds to an enzyme and reduces its activity.
• This prevents the enzyme from interacting with its substrate and, therefore, prevents the substrate from being converted into a product.

Types of Inhibition

• Competitive Inhibition: The inhibitor binds to the active site, preventing the substrate from binding.

• Uncompetitive Inhibition: The inhibitor binds only to the enzyme-substrate complex, preventing it from converting the substrate to a product.

• Noncompetitive Inhibition: The inhibitor can bind to either the enzyme or the enzyme-substrate complex, but not the active site. This reduces the overall enzyme activity.

Rate Equations

• The rate equations describe how the rate of a reaction affected by factors such as enzyme concentration, substrate concentration, and the presence of an inhibitor.

Enzyme Immobilization

• Enzyme immobilization is the process of attaching enzymes to an insoluble support medium or encapsulating them.

• This restricts the enzyme's movement.

• Advantages:

  • Reusable enzymes
  • Facilitates continuous operations.
  • Easier enzyme/product recovery
  • More stable enzymes
  • Facilitates process control of product yield and quality

• Methods:

  • Adsorption: Enzymes are physically attached to a support.
  • Entrapment: Enzymes are enclosed in a porous matrix.
  • Cross-linking: Enzymes are covalently bonded to another molecule to form a more stable complex.
  • Covalent binding: Enzymes are directly attached to a support material through covalent bonds.

Ideal Carrier/Support Specifications

• Inert
• Cheap
• Physically strong and stable
• Reduces product inhibition
• Discourages microbial growth
• Discourages non-specific adsorption

Entrapment Technique

• Common support material: polyacrylamide, calcium alginate, gelatin.

• Suitable for low molecular weight substrates and products.

• Advantages:

  • Enzymes are not chemically modified.
  • Enzyme properties are not altered.

• Disadvantages:

  • Deactivation of the enzyme may occur during gel formation.
  • Enzyme leakage may occur based on the support material's pore size. -Diffusional limitations may occur, reducing the accessibility of the substrate.

Microencapsulation

• Enzymes are entrapped in semi-permeable/microscopical membranes.

• Not suitable for proteolytic enzymes or macro-molecular substrates.

Cross-linking

• Covalent bonds are formed between enzyme molecules using multifunctional reagents, creating a 3-dimensional structure.

• Used to stabilize adsorbed enzymes and prevent leakage from materials like polyacrylamide gels

Covalent Binding

• The enzyme is bonded to the surface through covalent bond formation using specific functional groups.

Most Commonly Used Functional Groupings

• Amino groups
• Carboxyl groups
• Hydroxyl groups
• Sulphydryl groups

Advantages (Covalent Binding)

• Very strong bonding (less enzyme leakage)
• Small amounts of enzymes are immobilized
• Provides more permanent linkage between the enzyme and support material

Water-Insoluble Support Materials

• Synthetic: acrylamide-based polymers, maleic anhydride polymers, styrene-based polymers
• Natural: agarose, cellulose, dextran, glass, starch

Adsorption

• Oldest method; involves the physical attachment of enzymes to a surface via weak physical forces (van der Waals forces, dispersion forces)

• Advantages:

  • Easy and simple immobilization procedure.
  • Reversible adsorption process.
  • Enzymes are not deactivated. -Separation and purification of the enzymes possible during immobilization.

• Disadvantages:

  • Many other substances can be attached alongside immobilized enzymes due to the nonspecific nature of adsorption.
  • Loading of enzyme into limited amounts of surface area is low.
  • Weak bonding strength.

Materials for Adsorption

• Silica gel
• Metal oxides
• Glass
• Organic polymers
• Porous carbon
• Clay

Microbial Control

• Sterilization: Removal/destruction of all microbes (including endospores).
• Aseptic: Environment/procedure free of pathogens.
• Disinfection: Use of physical/chemical agents (disinfectants) to treat inanimate objects.
• Antisepsis: Using chemical/antimicrobial agents on skin/tissues.
• Degerming: Removal of microbes from a surface using rubbing.
• Sanitizing: Disinfecting places/utensils used by public to reduce pathogenic microbes and meet public health standards.
• Pasteurization: Use of heat to kill pathogens and reduce spoilage microorganisms in food/beverages.

Microbial Death

• Permanent termination of an organism's vital processes.
• Factors affecting death rate:
  • Number of microorganisms
  • Nature of microorganisms.
  • Temperature & pH of the environment.
  • Concentration of the agent
  • Mode of action of the agent
  • Presence of solvents/interfering organic matter/inhibitors
• Cellular targets of physical/chemical agents: cell wall, cell membrane, cellular synthetic processes (DNA/RNA), proteins.

Methods of Physical Control

• Heat
• Radiation
• Filtration
• Ultrasonic waves
• Cold

Heat

• Moist heat: Hot water, boiling water, steam (vaporized water); 60–135°C
• Dry heat: Heated by flame or electric coil; 160°C+

Radiation

• Types: Ionizing (x-rays, gamma rays) - creates free radicals destroying microbial proteins and DNA -Ultraviolet (UV) - damages nucleic acids by binding adjacent thymine bases; microbe dies

Filtration

• Modern microbial filters use cellulose acetate, polycarbonate, plastic materials (teflon & nylon); pores vary from coarse (8 microns) to ultrafine (0.02 microns)

Chemical Agents

• Gases for sterilization: ethylene oxide (ETO), beta propiolactone (BPL), formaldehyde
• Qualities in choosing antimicrobial chemical agents:
  • Rapid action in low concentration
  • Solubility in water/alcohol and long-term stability
  • Broad-spectrum microbicidal action without being toxic to human/animal tissues
  • Penetrates inanimate surfaces for cumulative/persistent action
  • Resistance to inactivation by organic matter
  • Non-corrosive/non-staining properties
  • Sanitizing and deodorizing properties
  • Affordability & ready availability

Fermentation

• Biochemical process using microorganisms on a substrate in the presence of nutrients to produce metabolic products.
• Factors for Success: Microorganism, medium, fermenter, nutrients/additives, continuous vs batch process.
• Issues/Challenges: Contamination, contaminated product outweighing desired product (especially in continuous fermentation), interfering with product recovery, unstable air presence resulting in spoilage.
• Solutions: Sterilize medium/inoculum/fermenter/pipes/valves/materials/air, aseptic conditions, maintain desired pH, use appropriate filters.

Sterilization of Medium, Air and Fermenters

• Methods: Boiling in water, passing steam, autoclaving (pressurized steam).
• Scale Processes: Adjusting pH, using contamination inhibitors (like lactic acid).
• Sensitive Enzymes/Nutrients: Separate initially, sterilize separately using bacterial filtration, add back to medium.
• Air Sterilization: Heating, UV rays, germicidal sprays, filtration (smaller/bigger pores)
• Fermenter Sterilization: Steam (15 PSIG) for 20 minutes, followed by flushing with sterile air.

Biochemical Engineering

• Applied chemical engineering principles using biological catalysts to desired chemical transformation, conducting biological processes on an industrial scale.
• Role: Biochemical engineers work with biological scientists to design reactors/processes, using the most suitable biological catalyst under optimal environmental condition for desired chemical transformations and optimal separation of the product from the mixture in the most economic way.

Biological Processes

• Main advantages: Mild reaction condition, specificity, effectiveness, renewable resources, recombinant DNA technology.
• Main disadvantages: Complex product mixtures, dilute aqueous environments, contamination, variability.

What Does Life Really Look Like?

• It discusses the history of microbiology.

• It mentiones the father of microbiology, Antonie van Leeuwenhoek.

Classification of Living Organisms

• Kingdom Monera (bacteria)
• Kingdom Protista (protozoa, algae)
• Kingdom Fungi (yeast, mushrooms)
• Kingdom Plantae (plants)
• Kingdom Animalia (animals)

Biological Basics

• Cells are the basic units of life.
• Prokaryotes: Simple cells (no nucleus or membrane-bound organelles).
• Eukaryotes: Complex cells (having a nucleus and membrane-bound organelles).
• Chemical composition: Water, trace elements, proteins, nucleic acids, lipids.
• Microbial Diversity: Psychrophile, Mesophile, Thermophile; Aerobic, Anaerobic, Facultative.

Derivation of Michaelis-Menten Model

• The model derives the rate of product formation and substrate consumption based on the enzyme-substrate complex assuming the product-releasing step to be the slower reaction rate and negligible change in the intermediate concentration.
• It emphasizes deriving the rate equation using the Michaelis-Menten and Briggs-Haldene approaches showing how the latter simplifies to the former if the product releasing step reaction rate is much slower than the enzyme-substrate dissociation step.