Lecture 1:
▪ recall one definition of what is biocatalysis
Biocatalysis is a chemical process where enzymes catalyze reactions between organic
compounds
▪ name the different types of biocatalysts and describe their differences
There are Ribsozymes, Abzymes, chemzymes and designer enzymes.
Ribozymes are used for cleaving RNA
Abzymes are used for stabilizing a transition state
Chemzymes are artificial tailored enzymes to catalyze specific reactions
Designer enzymes are for binding and stabilizing transition states
▪ name up to three examples for applications of enzymes in industry
Producing ethanol, plastics and pharmaceuticals
▪ discuss the advantages and disadvantages of biocatalysis
Biocatalysis is a technique that can catalyze a very broad range of reactions, its a green
process, efficient and its engineerable. Some downsides is that BC can only use one
enantiomer, there are risks of inhibition, only available for certain conditions (pH, solvent,
etc) and usually has the highest effectivity in a water or buffer solution.
▪ demonstrate the contribution of biocatalysis to the green chemistry principles
Biocatalysis contributes to 10 out of the 12 green chemistry principles
Lecture 2:
▪ describe the different methods to produce enantiopure compounds
Resolution of racemates with crystallization methods or kinetic resolution, Asymmetric
synthesis by converting a non chiral molecule to only 1 enantiomer, and exploitation of the
chiral pool by using enatiomers from nature.
▪ name the main differences between kinetic resolutions and dynamic kinetic
resolutions
In kinetic resolution you use a fast enzyme to convert the enantiomer into a product and a
slow enzyme to convert the ‘wrong’/other enantiomer into a product. This results in a yield of
50%. Dynamic kinetic resolution works similar but in this method the enantiomers can also
be converted into each other with an enzyme, resulting in a theoretical yield of 100%.
▪ explain the differences between substrate, product and catalytic promiscuity in
biocatalysis
Substrate promiscuity refers to when multiple substrates can be used by the enzyme to
result in different product. Product promiscuity refers to the situation where conversion of
one substrate can give multiple different products. Catalytic promiscuity refers to where an
enzyme can catalyze multiple different reactions.
1) What is the contribution of biocatalysis to the 12 Green Chemistry principles?
Biocatalysis contributes to 10 of the 12 green chemistry principles. It doesnt conform with the
principles that discuss the design of the product rather than the process.
2) How can you synthesize enantiomeric pure compounds?
By either using resolution of racemates by crystallization or kinetic resolution, Asymmetric
resolution by converting a non chiral molecule into only 1 enatiomer or by exploiting the
natural chiral pool and just using enantiomers in nature.
3) How can we make use of enzyme promiscuity for organic synthesis?
By utelizing enzyme promiscuity it is possible to broaden the spectrum over which the
enzyme can work, increasing productivity and efficiency.
▪ What is ee and E (enantiomeric excess)?
, ee refers to how pure a mixture of enantionmers is. When one enantiomer is 70% present
and the other is 30% present the ee of the mixture is (70-30=) 40%.
Lecture 3:
▪ name the main features of enzymes that can be targeted by protein engineering
Activity, productivity, efficiency and selectivity
▪ compare the main differences between rational protein design and directed
evolution
Directed evolution is randomized mutations that ultimately result in a very large library of
mutations that are screened or selected to pick the desired mutation. This method does not
require any knowledge of the structure of the protein to make this method work but the
outcome is all over the place. Rational protein design is much more precise method that
requires extensive knowledge about the 3d structure and sequences of the protein to very
precisely with site directed mutagenesis induce desired mutations.
▪ describe the importance of suitable screening and selection procedures in directed
evolution
In longer proteins the amount of different mutations that can happen can go into the millions,
meaning is it of importance to test what all the different mutations do. This is done with the
screening and selection methods
▪ describe the importance of using true vs. surrogate substrates in library screening
By utelizing surrogate substrates it will become way easier to do the library screening since
the change of substrate will also result in certain changes in the product. The product might
have a different colour or could be detectable under UV light. This significantly simplifies the
screening process.
Lecture 4:
▪ understand important methodologies in directed evolution (such as epPCR and site-
saturation mutagenesis) and rational protein design (such as QuikChange)
Error prone PCR works by doing PCR but with altered conditions. For instance instead of
using Mg you can use Mn, you can use different dNTP concentrations and a polymerase
without proof reading abilities. This raises the error rate to about 1-2%, causing mutations all
over.
Gene site saturation mutagenesis (GSSM) works by designing certain primers that are all
identical except for every primer to contain 1 different aa in a certain position. When
unleashing the primers on the sequence you want to implement the mutation in you can very
specifically change a single aa in the sequence and see what consequence this has.
▪ describe a comprehensive biocatalyst evolution to withstand harsh process
conditions
By using directed evolution to induce a large library of mutations, then using selection with
harsh conditions to see which strains are able to withstand the conditions and then
engineering/reapeting until the desired result is reached is a very easy and fast way to
evolve a biocatalyst.
Lecture 5:
• understand the difference between small molecule drugs and biologics.
Small molecule durgs are only up to 40 peptides and biologics are a bit larger than 40
peptides.
▪ recall one definition of what is biocatalysis
Biocatalysis is a chemical process where enzymes catalyze reactions between organic
compounds
▪ name the different types of biocatalysts and describe their differences
There are Ribsozymes, Abzymes, chemzymes and designer enzymes.
Ribozymes are used for cleaving RNA
Abzymes are used for stabilizing a transition state
Chemzymes are artificial tailored enzymes to catalyze specific reactions
Designer enzymes are for binding and stabilizing transition states
▪ name up to three examples for applications of enzymes in industry
Producing ethanol, plastics and pharmaceuticals
▪ discuss the advantages and disadvantages of biocatalysis
Biocatalysis is a technique that can catalyze a very broad range of reactions, its a green
process, efficient and its engineerable. Some downsides is that BC can only use one
enantiomer, there are risks of inhibition, only available for certain conditions (pH, solvent,
etc) and usually has the highest effectivity in a water or buffer solution.
▪ demonstrate the contribution of biocatalysis to the green chemistry principles
Biocatalysis contributes to 10 out of the 12 green chemistry principles
Lecture 2:
▪ describe the different methods to produce enantiopure compounds
Resolution of racemates with crystallization methods or kinetic resolution, Asymmetric
synthesis by converting a non chiral molecule to only 1 enantiomer, and exploitation of the
chiral pool by using enatiomers from nature.
▪ name the main differences between kinetic resolutions and dynamic kinetic
resolutions
In kinetic resolution you use a fast enzyme to convert the enantiomer into a product and a
slow enzyme to convert the ‘wrong’/other enantiomer into a product. This results in a yield of
50%. Dynamic kinetic resolution works similar but in this method the enantiomers can also
be converted into each other with an enzyme, resulting in a theoretical yield of 100%.
▪ explain the differences between substrate, product and catalytic promiscuity in
biocatalysis
Substrate promiscuity refers to when multiple substrates can be used by the enzyme to
result in different product. Product promiscuity refers to the situation where conversion of
one substrate can give multiple different products. Catalytic promiscuity refers to where an
enzyme can catalyze multiple different reactions.
1) What is the contribution of biocatalysis to the 12 Green Chemistry principles?
Biocatalysis contributes to 10 of the 12 green chemistry principles. It doesnt conform with the
principles that discuss the design of the product rather than the process.
2) How can you synthesize enantiomeric pure compounds?
By either using resolution of racemates by crystallization or kinetic resolution, Asymmetric
resolution by converting a non chiral molecule into only 1 enatiomer or by exploiting the
natural chiral pool and just using enantiomers in nature.
3) How can we make use of enzyme promiscuity for organic synthesis?
By utelizing enzyme promiscuity it is possible to broaden the spectrum over which the
enzyme can work, increasing productivity and efficiency.
▪ What is ee and E (enantiomeric excess)?
, ee refers to how pure a mixture of enantionmers is. When one enantiomer is 70% present
and the other is 30% present the ee of the mixture is (70-30=) 40%.
Lecture 3:
▪ name the main features of enzymes that can be targeted by protein engineering
Activity, productivity, efficiency and selectivity
▪ compare the main differences between rational protein design and directed
evolution
Directed evolution is randomized mutations that ultimately result in a very large library of
mutations that are screened or selected to pick the desired mutation. This method does not
require any knowledge of the structure of the protein to make this method work but the
outcome is all over the place. Rational protein design is much more precise method that
requires extensive knowledge about the 3d structure and sequences of the protein to very
precisely with site directed mutagenesis induce desired mutations.
▪ describe the importance of suitable screening and selection procedures in directed
evolution
In longer proteins the amount of different mutations that can happen can go into the millions,
meaning is it of importance to test what all the different mutations do. This is done with the
screening and selection methods
▪ describe the importance of using true vs. surrogate substrates in library screening
By utelizing surrogate substrates it will become way easier to do the library screening since
the change of substrate will also result in certain changes in the product. The product might
have a different colour or could be detectable under UV light. This significantly simplifies the
screening process.
Lecture 4:
▪ understand important methodologies in directed evolution (such as epPCR and site-
saturation mutagenesis) and rational protein design (such as QuikChange)
Error prone PCR works by doing PCR but with altered conditions. For instance instead of
using Mg you can use Mn, you can use different dNTP concentrations and a polymerase
without proof reading abilities. This raises the error rate to about 1-2%, causing mutations all
over.
Gene site saturation mutagenesis (GSSM) works by designing certain primers that are all
identical except for every primer to contain 1 different aa in a certain position. When
unleashing the primers on the sequence you want to implement the mutation in you can very
specifically change a single aa in the sequence and see what consequence this has.
▪ describe a comprehensive biocatalyst evolution to withstand harsh process
conditions
By using directed evolution to induce a large library of mutations, then using selection with
harsh conditions to see which strains are able to withstand the conditions and then
engineering/reapeting until the desired result is reached is a very easy and fast way to
evolve a biocatalyst.
Lecture 5:
• understand the difference between small molecule drugs and biologics.
Small molecule durgs are only up to 40 peptides and biologics are a bit larger than 40
peptides.
