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 (C)</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 (C)</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 (B)</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 (C)</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 (D)</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 (D)</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 (D)</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 (C)</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 (C)</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 (D)</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 (C)</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 (C)</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 (B)</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 (A)</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 (D)</p> Signup and view all the answers

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

<p>Error-prone PCR (C)</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 (B)</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 (A)</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 (B)</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 (C)</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 (A)</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 (C)</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 (B)</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 (A)</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 (C)</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 (A)</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 (B)</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 (C)</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 (D)</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 (C)</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 (B)</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 (C)</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 (B)</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 (C)</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 (B)</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 (D)</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 (D)</p> Signup and view all the answers

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