chapter 2 Factors Affecting Enzyme Engineering Success
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Questions and Answers

What is a critical factor in the success of enzyme engineering projects?

  • The availability of a suitable starting point (correct)
  • The speed of the evolution process
  • The type of environmental challenges
  • The number of mutations introduced simultaneously

    • What does a highly evolvable enzyme mean?

  • It cannot retain its overall structure with mutations
  • It can only accept one type of mutation
  • It can accept mutations while retaining its overall structure and catalytic function (correct)
  • It is completely resistant to mutations

    • What does robustness to mutations often correlate with?

  • Enzyme freezing
  • Configurational stability (correct)
  • Speed of evolution
  • Stability threshold

    • What is a downside of random directed evolution mentioned in the text?

    <p>Beneficial effect of a mutation can be concealed by the negative effect of other mutations</p> Signup and view all the answers

    What is critical for an enzyme to evolve a new function?

    <p>Ability to accept mutations while retaining its overall structure and catalytic function</p> Signup and view all the answers

    What is the consensus regarding enzymes' evolvability?

    <p>Enzymes are highly evolvable and can rapidly adapt to new functions</p> Signup and view all the answers

    What understanding can help engineers in their search for novel biocatalysts?

    <p>Understanding the limitations of enzyme evolution</p> Signup and view all the answers

    According to Maynard Smith, how must functional proteins form a network for natural selection to occur?

    <p>By forming a continuous network with unit mutational steps</p> Signup and view all the answers

    What happens in many directed evolution experiments?

    <p>They come to an early halt after only a minor improvement or no improvement at all</p> Signup and view all the answers

    Why must a foundation for divergence of the new function pre-exist in the enzyme superfamily?

    <p>To allow gradual and smooth formation of new functions</p> Signup and view all the answers

    What is the fundamental rule the author starts with for devising a directed evolution strategy suitable for enzymes?

    <p>You get what you screen for</p> Signup and view all the answers

    Which common microbes were used to produce libraries of mutant enzymes for testing?

    <p>Escherichia coli or yeast</p> Signup and view all the answers

    Why were the researchers limited to monitoring only a few thousand protein variants?

    <p>Constraints in expressing and arraying protein variants</p> Signup and view all the answers

    What kind of change in sequence would be expected to generate only small improvements in function?

    <p>One or two mutations away from the starting protein</p> Signup and view all the answers

    To achieve significant changes in enzyme function, what would the researchers have to do?

    <p>Multiply benefits over successive generations</p> Signup and view all the answers

    What is the purpose of mutagenesis in directed evolution of protein catalysts?

    <p>To optimize a protein's properties through iterative cycles of mutagenesis and screening</p> Signup and view all the answers

    What is the role of error-prone PCR in creating genetic diversity?

    <p>It introduces copying errors by imposing imperfect or 'sloppy' reaction conditions</p> Signup and view all the answers

    What is the purpose of screening or selection in directed evolution of protein catalysts?

    <p>To identify catalysts with improved properties</p> Signup and view all the answers

    Which technique is used to generate random variation in DNA in the laboratory?

    <p>Error-prone PCR</p> Signup and view all the answers

    What is required for an effective assay during directed evolution of protein catalysts?

    <p>Tight linkage of genotype and phenotype</p> Signup and view all the answers

    What is one key feature of engineer-able enzymes?

    <p>Their ability to retain their original activity while evolving towards new activity</p> Signup and view all the answers

    Why are generalist enzymes considered a great starting point for directed evolution?

    <p>Because they typically retain their original activity and exhibit wider promiscuity</p> Signup and view all the answers

    What does the text suggest is a useful way to compare potential starting enzymes for directed evolution?

    <p>Assessing their ability to use a range of alternative substrates</p> Signup and view all the answers

    What is a successful but rather laborious technique for amplifying an enzyme's promiscuity mentioned in the text?

    <p>Neutral drifting</p> Signup and view all the answers

    What is NOT guaranteed by promiscuity and/or broad substrate acceptance in the context of directed evolution?

    <p>High catalytic efficiency of the new enzyme</p> Signup and view all the answers

    What is the purpose of error-prone PCR in the context of directed evolution?

    <p>To introduce copying errors and generate random DNA variations</p> Signup and view all the answers

    What is the primary challenge in discovering protein sequences that provide new benefits?

    <p>Finding functional proteins adjacent to one another in sequence space</p> Signup and view all the answers

    What is critical for an effective assay during directed evolution of protein catalysts?

    <p>Tight linkage of genotype and phenotype</p> Signup and view all the answers

    What is the role of natural selection in finding sequences that retain function?

    <p>It filters and picks functional sequences from the vast sequence space</p> Signup and view all the answers

    How are catalysts with improved properties identified in the process of directed evolution?

    <p>By screening or selection</p> Signup and view all the answers

    How is fitness defined in the context of directed protein evolution?

    <p>By the performance based on artificial selection imposed</p> Signup and view all the answers

    What technique can be used to generate DNA libraries for enzyme variants in directed evolution?

    <p>Mutagenesis by PCR-methods</p> Signup and view all the answers

    What is the impact of directed evolution on the fitness landscape?

    <p>It creates a new fitness landscape with continuous paths to higher fitness</p> Signup and view all the answers

    What is the purpose of mutagenesis in directed evolution of protein catalysts?

    <p>To introduce changes in the encoding gene to generate DNA libraries</p> Signup and view all the answers

    What distinguishes directed evolution from natural evolution?

    <p>Directed evolution aims to make better proteins for specific purposes</p> Signup and view all the answers

    What is a fundamental rule the author starts with for devising a directed evolution strategy suitable for enzymes?

    <p>You get what you screen for</p> Signup and view all the answers

    Why were the researchers limited to monitoring only a few thousand protein variants?

    <p>Because they could only express and array a few thousand protein variants</p> Signup and view all the answers

    What is one key feature of engineer-able enzymes mentioned in the text?

    <p>They can yield large improvements in function with minimal changes in sequence</p> Signup and view all the answers

    What happens in many directed evolution experiments, according to the text?

    <p>Rare and only slightly improved protein mutants are deployed for screening assays</p> Signup and view all the answers

    What is required for an effective assay during directed evolution of protein catalysts, according to the text?

    <p>Reproducible screening assays capable of finding rare and slightly improved protein mutants</p> Signup and view all the answers

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