50 Questions
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
How has protein engineering impacted the timescale for realizing dramatic improvements in enzymatic performance?
The timescale has decreased
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
How did Codexis improve the activity and stability of the ketoreductase (KRED) mentioned in the text?
Used DNA shuffling
What was the initial issue with the enzyme activities mentioned in the text?
The enzyme activities were too low for commercial viability
How does protein engineering compare to natural evolution in terms of timescale mentioned in the text?
Protein engineering is faster than natural evolution
What is a decisive weakness of biocatalysts?
Limited stability with respect to temperature, solvents, pH value, ionic strength, and salt type
What was introduced by Smith and coworkers in 1985 as a method of rational design?
Rational design by site-directed mutagenesis (SDM)
What is the drawback of SDM as a method of rational design?
Requirement for detailed three-dimensional structure and mechanism information
What is the reason for the progress of biocatalysis over the past decade?
Advance of protein engineering
What does directed evolution require for enzyme engineering?
No structural information whatsoever
What is a major drawback associated with biocatalysts as a generally applicable technique?
Very limited range of stability with respect to temperature, solvents, pH value, ionic strength, and salt type
What is one of the major challenges that remain before the advantages of biocatalysis can be fully harnessed?
The integration of thermodynamics and biocatalytic process development
Why does changing 30-40 amino acids and screening tens of thousands of candidates require a large research team?
Because of the speed and complexity of the process
What sets an upper limit to reaction rates in biocatalysis?
Diffusion
What does enzyme engineering assume about individual mutations?
They are additive
What is highly desirable in designing new processes involving enzyme engineering?
A closer integration of thermodynamics and biocatalytic process development
Why is it still unclear which strategies are the better ones in enzyme engineering?
Because many engineering strategies will yield improved variants, but some will yield better variants and find them faster
What is a drawback of the computer-based design of new enzyme activities?
The designed enzymes often have low activity
What is a challenge in predicting the outcome of additional mutations in protein engineering?
Distinguishing between additive mutations and dead-end mutations
Why are the designed enzymes for catalyzing reactions not found in nature not practical for use?
The activities of these enzymes are currently too low
What is a limitation of the current computer-based design of enzymes?
The requirement for substantial further engineering
What is a result of recent advances in protein engineering?
Conversion of mouse proteins into human proteins
Why is better understanding of enzymatic catalysis dynamics said to be needed?
To achieve practical use of designed enzymes for new reactions
What was the purpose of Frances Arnold's 1993 paper?
To describe the use of errorprone PCR for random mutagenesis of enzymes
What did Stemmer report in 1994 as a complementary technique to errorprone PCR?
DNA shuffling
What did Reetz demonstrate in 1997 as a potential use of similar strategies to directed evolution?
Increasing the (enantio)selectivity of an enzyme
Why does directed evolution require high-throughput screening equipment?
To process huge libraries of variants
What is the aim of semi-rational engineering, which gained momentum since 2005?
To limit randomization to crucial positions in enzymes
How can in silico analysis help in semi-rational engineering?
By finding relevant hotspots for mutagenesis through structure and sequence analysis
What is the primary criterion used to gauge process-relevant activity of enzymes?
Enzyme specificity
At what substrate concentration is the enzyme specificity (kcat/KM) misleading for most industrial applications?
[S] > KM
What is the measure of binding affinity of the substrate to the enzyme in the Michaelis-Menten equation?
kcat/KM
What is the maximum number of possible combinations for protein sequences?
20N
Which class of macromolecules consists of a linear chain of 20 different types of amino acids?
Proteins
What is the range of amino acid residues in the monomers of enzymes mentioned in the text?
147 to 667
What is a decisive weakness of biocatalysts?
Limited range of stability with respect to temperature, solvents, pH value, ionic strength, and salt type
What was the arguably most important reason for the progress of biocatalysis over the past decade?
Advancement of protein engineering
What does rational design by site-directed mutagenesis (SDM) involve?
Introduction of point mutations with detailed information about the three-dimensional structure and mechanism of the enzyme
What does directed evolution require for enzyme engineering?
No structural information whatsoever
What is a major drawback of the computer-based design of new enzyme activities?
The designed enzymes have low activity
What sets an upper limit to reaction rates in biocatalysis?
The designed enzymes for catalyzing reactions not found in nature
Why does protein engineering still require testing 10–20 predictions for new enzyme activities?
Because computer design of new enzyme activities is not accurate
What is a significant drawback associated with biocatalysts as a generally applicable technique?
Limited range of stability with respect to temperature, solvents, pH value, ionic strength, and salt type
What is one of the most important reasons for the progress of biocatalysis over the past decade?
The advance of protein engineering
What does rational design by site-directed mutagenesis (SDM) involve?
Introduction of point mutations in the protein by replacing one amino acid with another
What is a drawback of directed evolution as a method for enzyme engineering?
It relies on chance rather than a set of generally applicable rules
What is the result of the engineered enzymes' changes in properties mentioned in the text?
They have improved catalytic properties quantitatively by factors of thousands to millions.
What is a particularly noteworthy example of biocatalysis mentioned in the text?
The synthesis of a key intermediate for atorvastatin, the active ingredient of Pfizer's cholesterol-lowering drug Lipitor.
What is a significant impact of protein engineering according to the text?
A dramatic improvement in enzymatic performance within a few months.
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.
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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