Chapter 1 Protein Engineering Techniques Quiz
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Questions and Answers

What is one of the significant outcomes of protein engineering mentioned in the text?

  • Improved catalytic properties of enzymes by factors of thousands to millions (correct)
  • No change in the catalytic properties of enzymes
  • Reduced stability of the engineered enzymes
  • Decreased catalytic properties of enzymes by factors of thousands to millions

    • How has protein engineering impacted the timescale for realizing dramatic improvements in enzymatic performance?

  • The timescale has remained the same
  • The timescale has become unpredictable
  • The timescale has increased
  • The timescale has decreased (correct)

    • What is an example of using biocatalysis as an important tool in chemical synthesis?

  • The two-step, three enzyme process for the synthesis of Lipitor (correct)
  • The three-step, two enzyme process for the synthesis of Lipitor
  • The one-step, three enzyme process for the synthesis of Lipitor
  • The two-step, four enzyme process for the synthesis of Lipitor

    • How did Codexis improve the activity and stability of the ketoreductase (KRED) mentioned in the text?

    <p>Used DNA shuffling</p> Signup and view all the answers

    What was the initial issue with the enzyme activities mentioned in the text?

    <p>The enzyme activities were too low for commercial viability</p> Signup and view all the answers

    How does protein engineering compare to natural evolution in terms of timescale mentioned in the text?

    <p>Protein engineering is faster than natural evolution</p> Signup and view all the answers

    What is a decisive weakness of biocatalysts?

    <p>Limited stability with respect to temperature, solvents, pH value, ionic strength, and salt type</p> Signup and view all the answers

    What was introduced by Smith and coworkers in 1985 as a method of rational design?

    <p>Rational design by site-directed mutagenesis (SDM)</p> Signup and view all the answers

    What is the drawback of SDM as a method of rational design?

    <p>Requirement for detailed three-dimensional structure and mechanism information</p> Signup and view all the answers

    What is the reason for the progress of biocatalysis over the past decade?

    <p>Advance of protein engineering</p> Signup and view all the answers

    What does directed evolution require for enzyme engineering?

    <p>No structural information whatsoever</p> Signup and view all the answers

    What is a major drawback associated with biocatalysts as a generally applicable technique?

    <p>Very limited range of stability with respect to temperature, solvents, pH value, ionic strength, and salt type</p> Signup and view all the answers

    What is one of the major challenges that remain before the advantages of biocatalysis can be fully harnessed?

    <p>The integration of thermodynamics and biocatalytic process development</p> Signup and view all the answers

    Why does changing 30-40 amino acids and screening tens of thousands of candidates require a large research team?

    <p>Because of the speed and complexity of the process</p> Signup and view all the answers

    What sets an upper limit to reaction rates in biocatalysis?

    <p>Diffusion</p> Signup and view all the answers

    What does enzyme engineering assume about individual mutations?

    <p>They are additive</p> Signup and view all the answers

    What is highly desirable in designing new processes involving enzyme engineering?

    <p>A closer integration of thermodynamics and biocatalytic process development</p> Signup and view all the answers

    Why is it still unclear which strategies are the better ones in enzyme engineering?

    <p>Because many engineering strategies will yield improved variants, but some will yield better variants and find them faster</p> Signup and view all the answers

    What is a drawback of the computer-based design of new enzyme activities?

    <p>The designed enzymes often have low activity</p> Signup and view all the answers

    What is a challenge in predicting the outcome of additional mutations in protein engineering?

    <p>Distinguishing between additive mutations and dead-end mutations</p> Signup and view all the answers

    Why are the designed enzymes for catalyzing reactions not found in nature not practical for use?

    <p>The activities of these enzymes are currently too low</p> Signup and view all the answers

    What is a limitation of the current computer-based design of enzymes?

    <p>The requirement for substantial further engineering</p> Signup and view all the answers

    What is a result of recent advances in protein engineering?

    <p>Conversion of mouse proteins into human proteins</p> Signup and view all the answers

    Why is better understanding of enzymatic catalysis dynamics said to be needed?

    <p>To achieve practical use of designed enzymes for new reactions</p> Signup and view all the answers

    What was the purpose of Frances Arnold's 1993 paper?

    <p>To describe the use of errorprone PCR for random mutagenesis of enzymes</p> Signup and view all the answers

    What did Stemmer report in 1994 as a complementary technique to errorprone PCR?

    <p>DNA shuffling</p> Signup and view all the answers

    What did Reetz demonstrate in 1997 as a potential use of similar strategies to directed evolution?

    <p>Increasing the (enantio)selectivity of an enzyme</p> Signup and view all the answers

    Why does directed evolution require high-throughput screening equipment?

    <p>To process huge libraries of variants</p> Signup and view all the answers

    What is the aim of semi-rational engineering, which gained momentum since 2005?

    <p>To limit randomization to crucial positions in enzymes</p> Signup and view all the answers

    How can in silico analysis help in semi-rational engineering?

    <p>By finding relevant hotspots for mutagenesis through structure and sequence analysis</p> Signup and view all the answers

    What is the primary criterion used to gauge process-relevant activity of enzymes?

    <p>Enzyme specificity</p> Signup and view all the answers

    At what substrate concentration is the enzyme specificity (kcat/KM) misleading for most industrial applications?

    <p>[S] &gt; KM</p> Signup and view all the answers

    What is the measure of binding affinity of the substrate to the enzyme in the Michaelis-Menten equation?

    <p>kcat/KM</p> Signup and view all the answers

    What is the maximum number of possible combinations for protein sequences?

    <p>20N</p> Signup and view all the answers

    Which class of macromolecules consists of a linear chain of 20 different types of amino acids?

    <p>Proteins</p> Signup and view all the answers

    What is the range of amino acid residues in the monomers of enzymes mentioned in the text?

    <p>147 to 667</p> Signup and view all the answers

    What is a decisive weakness of biocatalysts?

    <p>Limited range of stability with respect to temperature, solvents, pH value, ionic strength, and salt type</p> Signup and view all the answers

    What was the arguably most important reason for the progress of biocatalysis over the past decade?

    <p>Advancement of protein engineering</p> Signup and view all the answers

    What does rational design by site-directed mutagenesis (SDM) involve?

    <p>Introduction of point mutations with detailed information about the three-dimensional structure and mechanism of the enzyme</p> Signup and view all the answers

    What does directed evolution require for enzyme engineering?

    <p>No structural information whatsoever</p> Signup and view all the answers

    What is a major drawback of the computer-based design of new enzyme activities?

    <p>The designed enzymes have low activity</p> Signup and view all the answers

    What sets an upper limit to reaction rates in biocatalysis?

    <p>The designed enzymes for catalyzing reactions not found in nature</p> Signup and view all the answers

    Why does protein engineering still require testing 10–20 predictions for new enzyme activities?

    <p>Because computer design of new enzyme activities is not accurate</p> Signup and view all the answers

    What is a significant drawback associated with biocatalysts as a generally applicable technique?

    <p>Limited range of stability with respect to temperature, solvents, pH value, ionic strength, and salt type</p> Signup and view all the answers

    What is one of the most important reasons for the progress of biocatalysis over the past decade?

    <p>The advance of protein engineering</p> Signup and view all the answers

    What does rational design by site-directed mutagenesis (SDM) involve?

    <p>Introduction of point mutations in the protein by replacing one amino acid with another</p> Signup and view all the answers

    What is a drawback of directed evolution as a method for enzyme engineering?

    <p>It relies on chance rather than a set of generally applicable rules</p> Signup and view all the answers

    What is the result of the engineered enzymes' changes in properties mentioned in the text?

    <p>They have improved catalytic properties quantitatively by factors of thousands to millions.</p> Signup and view all the answers

    What is a particularly noteworthy example of biocatalysis mentioned in the text?

    <p>The synthesis of a key intermediate for atorvastatin, the active ingredient of Pfizer's cholesterol-lowering drug Lipitor.</p> Signup and view all the answers

    What is a significant impact of protein engineering according to the text?

    <p>A dramatic improvement in enzymatic performance within a few months.</p> Signup and view all the answers

    Study Notes

    Protein Engineering Outcomes

    • One significant outcome of protein engineering is the dramatic improvement in enzymatic performance.
    • Protein engineering has impacted the timescale for realizing dramatic improvements in enzymatic performance, allowing for improvements in a matter of years, compared to millions of years in natural evolution.

    Biocatalysis in Chemical Synthesis

    • Biocatalysis is an important tool in chemical synthesis, with an example being the use of ketoreductase (KRED) for the production of chemicals.

    Improving Enzyme Activity and Stability

    • Codexis improved the activity and stability of the ketoreductase (KRED) by using protein engineering techniques.
    • The initial issue with the enzyme activities was that they were not suitable for industrial applications.

    Comparison to Natural Evolution

    • Protein engineering is significantly faster than natural evolution, with improvements achieved in years, compared to millions of years.

    Biocatalysts Weakness

    • A decisive weakness of biocatalysts is their limited applicability and scalability.

    Rational Design

    • Smith and coworkers introduced site-directed mutagenesis (SDM) as a method of rational design in 1985.
    • The drawback of SDM is that it requires a large research team and significant resources.

    Directed Evolution

    • Directed evolution requires high-throughput screening equipment and testing of tens of thousands of candidates.
    • A major drawback of directed evolution is the requirement for large research teams and significant resources.

    Progress of Biocatalysis

    • The reason for the progress of biocatalysis over the past decade is the development of protein engineering techniques.
    • A major challenge that remains before the advantages of biocatalysis can be fully harnessed is the need for better understanding of enzymatic catalysis dynamics.

    Enzyme Engineering

    • Directed evolution requires a large research team and significant resources.
    • Enzyme engineering assumes that individual mutations will have a predictable effect on enzyme activity.
    • Highly desirable in designing new processes involving enzyme engineering is the ability to predict the outcome of additional mutations.

    Limitations of Computer-Based Design

    • A limitation of the current computer-based design of enzymes is that it requires testing 10–20 predictions for new enzyme activities.
    • A drawback of the computer-based design of new enzyme activities is that it is not always effective.

    Semi-Rational Engineering

    • The aim of semi-rational engineering, which gained momentum since 2005, is to design new enzymes with improved activity and stability.
    • In silico analysis can help in semi-rational engineering by predicting the outcome of additional mutations.

    Enzyme Activity

    • The primary criterion used to gauge process-relevant activity of enzymes is the Michaelis-Menten equation.
    • The measure of binding affinity of the substrate to the enzyme in the Michaelis-Menten equation is KM.

    Proteins

    • Proteins consist of a linear chain of 20 different types of amino acids.
    • The range of amino acid residues in the monomers of enzymes is 50-100.
    • The maximum number of possible combinations for protein sequences is 20^100.

    Impact of Protein Engineering

    • A significant impact of protein engineering is the ability to design new enzymes with improved activity and stability.
    • A particularly noteworthy example of biocatalysis is the use of ketoreductase (KRED) for the production of chemicals.
    • The result of the engineered enzymes' changes in properties is the improvement in enzymatic performance.

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    Description

    Test your knowledge of protein engineering techniques, including methods for identifying cooperative effects, predicting additive mutations, and computer-based design of new enzyme activities. Learn about the challenges and limitations in protein engineering.

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