Enzyme Classification and Properties
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Questions and Answers

What is the primary source of enzymes for industrial production?

  • Microorganisms (correct)
  • Plants
  • Human body
  • Animals

What is the first step in enzyme purification?

  • Chromatography
  • Clarification
  • Crude extract preparation
  • Cell lysis/disruption (correct)

Which technique is NOT commonly used in enzyme purification?

  • Centrifugation
  • Electrophoresis
  • PCR amplification (correct)
  • Ultrafiltration

What type of chromatography separates proteins based on specific binding?

<p>Affinity chromatography (B)</p> Signup and view all the answers

Which of the following methods is a form of cell lysis?

<p>Sonication (A)</p> Signup and view all the answers

Ammonium sulfate precipitation is primarily used for what purpose in enzyme purification?

<p>Selective protein precipitation (B)</p> Signup and view all the answers

Which step follows crude extract preparation in a typical enzyme purification scheme?

<p>Ammonium sulfate precipitation (D)</p> Signup and view all the answers

What is the purpose of using polyacrylamide gel electrophoresis in enzyme purification?

<p>To analyze protein purity (A)</p> Signup and view all the answers

What is the main goal of the purification process described?

<p>To achieve high purity while maintaining enzyme activity (B)</p> Signup and view all the answers

Which purification technique is applied after ion exchange chromatography in the outlined process?

<p>Affinity chromatography on Blue Sepharose (A)</p> Signup and view all the answers

Which of the following enzymes is NOT listed as a therapeutic enzyme in the content?

<p>Thrombin (B)</p> Signup and view all the answers

What is a common use of enzymes in diagnostic applications?

<p>In ELISA for detecting antibodies or antigens (B)</p> Signup and view all the answers

What type of enzyme is used in enzyme replacement therapy for Gaucher disease?

<p>Alglucerase/imiglucerase (D)</p> Signup and view all the answers

Which enzyme is NOT typically used in drug manufacturing processes mentioned?

<p>Collagenase (A)</p> Signup and view all the answers

Which of the following therapeutic applications uses thrombolytic enzymes?

<p>Dissolving blood clots (D)</p> Signup and view all the answers

Which step in the purification process is aimed at separating proteins based on size?

<p>Gel filtration on Sephadex G-200 (B)</p> Signup and view all the answers

What is one primary purpose of using enzyme-activated prodrugs in drug delivery systems?

<p>To achieve targeted drug delivery (D)</p> Signup and view all the answers

Which of the following enzymes is used in enzyme replacement therapy for Gaucher disease?

<p>Alglucerase/imiglucerase (B)</p> Signup and view all the answers

What role do thrombolytic enzymes serve in cardiovascular disease treatment?

<p>To dissolve blood clots (C)</p> Signup and view all the answers

Which enzyme is primarily used for addressing lactose intolerance?

<p>Lactase (B)</p> Signup and view all the answers

What is a significant application of lysozyme in medical treatments?

<p>For antibacterial purposes (A)</p> Signup and view all the answers

What is the optimal temperature range for incubating the inoculated medium during fermentation?

<p>28-30°C (A)</p> Signup and view all the answers

What therapeutic property can be improved by modifying enzymes in protein engineering?

<p>Enhancing specificity (D)</p> Signup and view all the answers

Which enzyme is commonly utilized for the treatment of gout?

<p>Uricase (A)</p> Signup and view all the answers

Which method is NOT recommended for disrupting cells to extract intracellular enzymes?

<p>Centrifugation (A)</p> Signup and view all the answers

What is the main benefit of using immobilized enzyme technology in biosensors?

<p>To enhance enzyme stability and reusability (C)</p> Signup and view all the answers

What is the purpose of adding protamine sulfate during the nucleic acid removal step?

<p>To precipitate nucleic acids (A)</p> Signup and view all the answers

Which chromatography technique could be used to further purify enzymes beyond basic chromatography?

<p>Gel filtration chromatography (C)</p> Signup and view all the answers

During enzyme characterization, which of the following should NOT be measured?

<p>Color intensity (B)</p> Signup and view all the answers

What is the primary goal when monitoring enzyme activity and protein concentration throughout the enzyme production process?

<p>To track purification progress and yield (A)</p> Signup and view all the answers

If the enzyme produces excessive foam during fermentation, which aspect should be adjusted?

<p>Aeration and agitation (B)</p> Signup and view all the answers

When storing purified enzymes, what is a common temperature to maintain for stability?

<p>4°C or -20°C (B)</p> Signup and view all the answers

Which enzyme is commonly used for its antioxidant properties?

<p>Superoxide dismutase (D)</p> Signup and view all the answers

What is the suitable temperature range for the fermentation process of amylase production?

<p>28-30°C (D)</p> Signup and view all the answers

What is one of the initial steps in producing amylglucosidase?

<p>Strain Selection (C)</p> Signup and view all the answers

During amylglucosidase production, what should be added to achieve 80% saturation in the ammonium sulfate precipitation step?

<p>Ammonium sulfate (B)</p> Signup and view all the answers

In the enzyme extraction process, which buffer is recommended for homogenizing the fermented substrate?

<p>0.1 M acetate buffer, pH 4.8 (A)</p> Signup and view all the answers

What type of medium is often used for fermenting to produce amylglucosidase?

<p>Solid substrate medium (A)</p> Signup and view all the answers

What is the purpose of dialysis in the amylglucosidase production process?

<p>To remove excess salt (B)</p> Signup and view all the answers

At what point in the amylglucosidase production process is the crude enzyme extract stored at 4°C?

<p>After sifting through Whatman filter paper (B)</p> Signup and view all the answers

What are appropriate carbon sources for preparing the growth medium?

<p>Starch and maltose (C)</p> Signup and view all the answers

What is the typical pH range for adjusting the growth medium?

<p>6.5 - 7.5 (A)</p> Signup and view all the answers

What method is used to collect the supernatant containing extracellular amylase after fermentation?

<p>Centrifugation (D)</p> Signup and view all the answers

Which step involves adding solid ammonium sulfate to achieve 60-80% saturation?

<p>Ammonium sulfate precipitation (C)</p> Signup and view all the answers

What is the purpose of dialysis in the enzyme purification process?

<p>To remove excess salt (D)</p> Signup and view all the answers

Which chromatography method is used to separate enzymes based on their charge?

<p>Ion exchange chromatography (B)</p> Signup and view all the answers

What is measured during the enzyme assay to evaluate enzyme activity?

<p>Reducing sugars released (A)</p> Signup and view all the answers

At what temperature range should the medium typically be incubated during fermentation?

<p>30-50°C (A)</p> Signup and view all the answers

Flashcards

Enzyme Extraction

The process of removing enzymes from their source (e.g., cells).

Cell Lysis/Disruption

Breaking open cells to release intracellular enzymes.

Enzyme Purification

The process of separating and isolating a desired enzyme from other proteins and contaminants.

Chromatography (Enzyme Purification)

Separating enzymes based on their physical and chemical properties using column chromatography.

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Ammonium Sulfate Precipitation

A method of enzyme purification that uses ammonium sulfate to selectively precipitate proteins.

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Electrophoresis (Enzyme Purification)

Separating enzymes by size and charge using an electric field.

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Ultrafiltration

Purifying or concentrating enzymes using membranes to separate based on size.

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Crude Enzyme Extract

An initial mixture of enzymes and some other cell materials after the first step of extraction.

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Affinity chromatography

A purification technique using a specific molecule (ligand) bound to a resin to isolate a target protein.

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Therapeutic Enzymes

Enzymes used directly as drugs to treat diseases and conditions.

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Enzyme Replacement Therapy

Using enzymes to treat enzyme deficiency disorders.

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Diagnostic Applications (enzymes)

Enzymes used in medical tests to diagnose conditions like diabetes or liver disease.

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Drug Manufacturing (enzyme use)

Using enzymes to create or modify pharmaceutical compounds.

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L-Asparaginase

Enzyme used to treat acute lymphoblastic leukemia.

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Collagenase

Enzyme used for wound healing and burn treatment.

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Enzyme-activated prodrugs

Drugs that are inactive until activated by a specific enzyme in the body.

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Enzyme-responsive nanocarriers

Nanocarriers for drug delivery that are activated by specific enzymes, enabling targeted drug delivery.

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Immobilized Enzyme Technology

Enzymes attached to a surface or support for use in biosensors and bioreactors.

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Enzyme specificity

The ability of an enzyme to catalyze a particular reaction or to bind to a specific type of substrate.

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Enzyme activity

Measuring how much product an enzyme produces from a substrate.

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Therapeutic properties of enzymes (modification)

Enzymes are modified to improve their effectiveness in treating diseases.

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What are enzymes used for?

Enzymes are biological catalysts that speed up chemical reactions in living organisms. They are essential for many biological processes, such as digestion, metabolism, and DNA replication.

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Superoxide dismutase (SOD)

SOD is an enzyme that helps neutralize harmful free radicals called superoxide anions, which can damage cells and contribute to aging and disease.

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Catalase

Catalase is an enzyme that breaks down hydrogen peroxide, a toxic by-product of metabolism, into water and oxygen.

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How are enzymes used in medicine?

Enzymes are used in medicine as therapeutic agents, often to treat genetic disorders or common health issues. They are also used in diagnosis and as tools for medical research.

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Amyloglucosidase

Amyloglucosidase is an enzyme that breaks down starch into glucose. It is often used in the production of glucose syrups and other food products.

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What is the first step in producing amyloglucosidase?

The first step is to select a suitable microorganism, usually a fungus like Aspergillus niger, to produce the enzyme.

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What is the optimal temperature for amyloglucosidase production?

The optimal temperature for amyloglucosidase production is usually between 28-30°C. This is the ideal temperature range for the microorganisms to produce the enzyme efficiently.

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What is the purpose of ammonium sulfate precipitation?

Ammonium sulfate precipitation is a method used to purify enzymes by selectively separating them from other proteins. It works by adding ammonium sulfate to the crude enzyme extract, causing some proteins to precipitate out.

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Inoculation

Introducing a selected strain of microorganism into a sterilized growth medium to initiate fermentation.

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Fermentation Conditions

Maintaining optimal temperature (usually 28-30°C), proper aeration, and agitation during fermentation.

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Cell Disruption (Intracellular Enzymes)

Breaking open cells to release intracellular enzymes, usually using methods like sonication or homogenization.

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Protamine Sulfate

A reagent used to precipitate nucleic acids (DNA and RNA) from crude enzyme extract.

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Elution

Releasing the bound enzyme from the chromatography column by washing with a specific solution, often containing a salt gradient.

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Specific Activity

A measure of enzyme purity, defined as enzyme activity per unit of protein.

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Growth Medium for Amylase

A solution containing nutrients like starch (carbon source), peptone (nitrogen source), and mineral salts, carefully adjusted to the optimal pH (6.5-7.5) for amylase production.

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Inoculation & Fermentation

Introducing the selected microbial strain into the prepared growth medium and allowing it to grow and produce amylase under controlled conditions (temperature, aeration, agitation).

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Enzyme Extraction (Amylase)

Separating the amylase enzyme produced by the microbe from the growth medium using a centrifuge, which spins the mixture and collects the enzyme-containing liquid (supernatant).

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Crude Enzyme Preparation

Initial processing of the extracted enzyme solution, involving filtration to remove cell debris and potential concentration to increase the enzyme concentration.

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Dialysis (Enzyme Purification)

A technique used to remove excess salt (added during ammonium sulfate precipitation) from the purified enzyme solution by allowing it to diffuse through a semi-permeable membrane.

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Ion Exchange Chromatography

A method of separation based on the charge of molecules, where the enzyme is loaded on a column containing charged beads and eluted using a salt gradient, separating amylase from other proteins.

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Gel Filtration Chromatography

Further purification method where the enzyme mixture is passed through a column filled with porous beads, separating proteins by size, allowing amylase to be collected in specific fractions.

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Study Notes

Enzyme Classification

  • Enzymes are grouped by the type of reaction they catalyze
  • Six major groups:
    • Oxidoreductases (EC 1): catalyze oxidation-reduction reactions
    • Transferases (EC 2): catalyze the transfer of functional groups
    • Hydrolases (EC 3): catalyze hydrolysis reactions
    • Lyases (EC 4): catalyze non-hydrolytic addition or removal of groups from substrates
    • Isomerases (EC 5): catalyze intramolecular rearrangements
    • Ligases (EC 6): catalyze the joining of two molecules
  • Each enzyme is assigned a unique 4-digit EC number

General Properties of Enzymes

  • Catalytic activity: enzymes accelerate chemical reactions without being consumed
  • Lower activation energy of reactions
  • Most enzymes catalyze reactions in both directions
  • Specificity: enzymes are highly specific for their substrates
  • Protein nature: most enzymes are proteins, their activity depends on maintaining specific 3D structure
  • Temperature sensitivity: enzyme activity increases with temperature up to an optimum (around 37°C for humans)
  • pH sensitivity: each enzyme has an optimum pH at which its activity is highest
  • Cofactor requirements: many enzymes require non-protein components (cofactors) for activity, cofactors can be metal ions or organic molecules
  • Regulation: enzyme activity can be regulated by various mechanisms (allosteric regulation, feedback inhibition, covalent modification)
  • Saturation kinetics: enzyme-catalyzed reactions show saturation kinetics
  • Inhibition: enzyme activity can be inhibited by specific molecules (competitive, non-competitive, or uncompetitive inhibitors)
  • Turnover number: enzymes have high turnover numbers (typically 100-1000 reactions per second)

Key Aspects of Enzyme Dynamics

  • Protein flexibility and motion: enzymes are not rigid structures, they exhibit complex internal dynamic motions
  • Timescales of motion: enzyme dynamics occur across various timescales (femtoseconds to milliseconds)
  • Conformational changes: enzymes undergo conformational changes during catalysis (induced fit, conformational selection)

Electrostatic Fluctuations

  • Electrostatic potential fluctuations are a key dynamical factor in reactions involving large changes in bond polarity

Substrate Binding and Product Release

  • Conformational changes in enzymes facilitate substrate entry into the active site and product release

Experimental and Computational Approaches

  • X-ray crystallography
  • NMR spectroscopy
  • Molecular Dynamics Simulations
  • Enzyme Kinetics

Main Sources of Enzymes

  • Microorganisms (bacteria, fungi, yeast)
  • Plants
  • Animals
  • Human body

Enzyme Extraction and Purification

  • Cell lysis/disruption
  • Mechanical disruption (e.g., homogenization, sonication)
  • Chemical lysis (using detergents or enzymes)
  • Clarification (removing cell debris)
  • Precipitation
  • Chromatography (ion exchange, size exclusion, affinity)
  • Electrophoresis
  • Ultrafiltration
  • Crystallization

Therapeutic Enzyme Applications

  • L-Asparaginase for leukemia treatment
  • Collagenase for wound healing
  • Digestive enzymes for digestive disorders
  • Thrombolytic enzymes for dissolving blood clots
  • Enzyme replacement therapy (e.g., for lysosomal storage diseases, Gaucher disease, Fabry disease)

Diagnostic Enzyme Applications

  • ELISA (Enzyme-Linked Immunosorbent Assay) for detecting antibodies or antigens
  • Glucose oxidase in diabetes testing
  • Alkaline phosphatase and other enzymes in liver function tests

Drug Manufacturing Applications

  • Biocatalysts in pharmaceutical compound production (e.g., penicillin acylase for antibiotics)
  • Lipases and esterases for chiral drug synthesis
  • Cytochrome P450 for drug metabolism
  • Drug delivery systems (enzyme-activated prodrugs, enzyme-responsive nanocarriers)

Other Applications

  • Analytical applications (determining substrate concentrations and enzyme activities)
  • Protein engineering (modifying enzyme properties)
  • Synthetic biology (designing artificial enzymes)
  • Immobilized enzyme technology (use in biosensors, bioreactors)

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Related Documents

Enzyme Classification & Properties PDF

Description

Explore the classification of enzymes based on the reactions they catalyze, including the six major groups such as oxidoreductases and transferases. Additionally, understand general properties of enzymes, including their catalytic activity and specificity. This quiz will enhance your knowledge of enzymatic functions and characteristics.

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