Advance biocatalysis – JJ 24
Advanced biocatalysis
Inhoud
Lecture 2: Biocatalytic application ...........................................................................................2
Enzyme catalysis .................................................................................................................4
Enzyme kinetics ...................................................................................................................8
Biocatalysis source ............................................................................................................ 15
Lecture 3: Costs of a Biocatalyst ............................................................................................ 16
Solvents & biocatalysis ...................................................................................................... 16
Immobilization................................................................................................................... 17
Discovery of an oxidase ...................................................................................................... 20
Lecture 4: Directing Engineering ............................................................................................. 21
Lecture 5: Hydrolases ............................................................................................................ 25
Lecture 6: Cofactors .............................................................................................................. 37
Lecture 7: Flavin metals ......................................................................................................... 49
Lecture 8: Enzymatic polymerization ...................................................................................... 58
Lecture 9: Enzymatic Polymer degradation ............................................................................. 61
1
,Advance biocatalysis – JJ 24
Lecture 2: Biocatalytic application
Regioselectivity: steroid hydroxylation
Hydrocortisone: anti-inflammatory agent
➢ Synthesized from plant precursors: stigmasterol
➢ Converted chemically to cortodoxone
➢ Regio and stereoselective steroid hydroxylation: 11-β-position
Chemical approaches combined with micro-organism/enzyme
Look for a molecule that is available in nature
Hydroxylation is quite difficult for steroids and having it
enantiomer pure as well
o Screen a lot of micro-organisms and find the proper
enzyme
Environmental and cost advantages: penicillin hydrolysis
➢ Penicillin G by fermentation
➢ Penicillin acylase is used for the production of 6-APA
➢ Intermediate for semi-synthetic antibiotics
➢ Compared to chemical cleavage
o Reduced use of solvents
o Fewer steps, cheaper
Often done for antibiotics
o Use penicillin → but quite fast resistance was
developed, so nowadays used chemically modified
penicillin-based antibiotic
6-amino-penicillanic acid is often used as a starting
compound for various antibiotics
Enzymes can hydrolyze the bond and form the amide (?)
Acrylamide
➢ For polyacrylamide
➢ High-molecular coagulant, paper reinforcement material,
adhesives
➢ Classical: copper catalyst
Formed chemically, but now an enzyme is used → cell-based process that promoted the
enzyme
With chemical processes, there were always some traces of copper present
Biocatalysis: no copper traces anymore, and the same costs
Replacing copper + H2SO4 catalyst
2
,Advance biocatalysis – JJ 24
Atorvastatin
Top selling drug (cardiovascular disease)
Enzymes able to act on …hydrines
Lower blood pressure
Cardiovascular diseases
Start: ketone → reduction → incorporate in the final product
KRED: ketone reductase
HHDH: halohylindehydronase
GDH: glucose dehydrogenase
ee has to be higher than 99.5% to be relevant
Sitagliptin
↳Anti-diabetic medication
↳Very powerful enzyme that
converts ketone to correct amine.
Transaminase
o Outperforms
chemical process
Screened transaminases
Biocatalytic cascade to islatravir
Nine-enzyme cascade to produce HIV drug islatravir
o No purification of intermediates was necessary
o Immobilized enzymes shown attached to spheres.
One pot reaction, but in different steps they put different enzymes
3
, Advance biocatalysis – JJ 24
Molunupiravir
Molecule active helping patients to survive COVID-19
Cascade reaction
Enzyme catalysis
- Study chemical process
- Mathematical skills so study kinetics
o Most important
o Increase rate
▪ Transition state
stabilization
- Physics, understanding the forces
- Biology, because complicated
structures activate by..
➢ Increase rate of reaction
o Transition state stabilization
➢ Do not change the equilibrium point of reactions
o Cascade to overcome equilibrium issues
➢ Specific for their substrate, active-site geometry, electrostatics
o Hydrophobic interactions (van der Waals)
o Polar (H-bonds)
o Ionic interactions (salt bridges)
o Induced fit
4
Advanced biocatalysis
Inhoud
Lecture 2: Biocatalytic application ...........................................................................................2
Enzyme catalysis .................................................................................................................4
Enzyme kinetics ...................................................................................................................8
Biocatalysis source ............................................................................................................ 15
Lecture 3: Costs of a Biocatalyst ............................................................................................ 16
Solvents & biocatalysis ...................................................................................................... 16
Immobilization................................................................................................................... 17
Discovery of an oxidase ...................................................................................................... 20
Lecture 4: Directing Engineering ............................................................................................. 21
Lecture 5: Hydrolases ............................................................................................................ 25
Lecture 6: Cofactors .............................................................................................................. 37
Lecture 7: Flavin metals ......................................................................................................... 49
Lecture 8: Enzymatic polymerization ...................................................................................... 58
Lecture 9: Enzymatic Polymer degradation ............................................................................. 61
1
,Advance biocatalysis – JJ 24
Lecture 2: Biocatalytic application
Regioselectivity: steroid hydroxylation
Hydrocortisone: anti-inflammatory agent
➢ Synthesized from plant precursors: stigmasterol
➢ Converted chemically to cortodoxone
➢ Regio and stereoselective steroid hydroxylation: 11-β-position
Chemical approaches combined with micro-organism/enzyme
Look for a molecule that is available in nature
Hydroxylation is quite difficult for steroids and having it
enantiomer pure as well
o Screen a lot of micro-organisms and find the proper
enzyme
Environmental and cost advantages: penicillin hydrolysis
➢ Penicillin G by fermentation
➢ Penicillin acylase is used for the production of 6-APA
➢ Intermediate for semi-synthetic antibiotics
➢ Compared to chemical cleavage
o Reduced use of solvents
o Fewer steps, cheaper
Often done for antibiotics
o Use penicillin → but quite fast resistance was
developed, so nowadays used chemically modified
penicillin-based antibiotic
6-amino-penicillanic acid is often used as a starting
compound for various antibiotics
Enzymes can hydrolyze the bond and form the amide (?)
Acrylamide
➢ For polyacrylamide
➢ High-molecular coagulant, paper reinforcement material,
adhesives
➢ Classical: copper catalyst
Formed chemically, but now an enzyme is used → cell-based process that promoted the
enzyme
With chemical processes, there were always some traces of copper present
Biocatalysis: no copper traces anymore, and the same costs
Replacing copper + H2SO4 catalyst
2
,Advance biocatalysis – JJ 24
Atorvastatin
Top selling drug (cardiovascular disease)
Enzymes able to act on …hydrines
Lower blood pressure
Cardiovascular diseases
Start: ketone → reduction → incorporate in the final product
KRED: ketone reductase
HHDH: halohylindehydronase
GDH: glucose dehydrogenase
ee has to be higher than 99.5% to be relevant
Sitagliptin
↳Anti-diabetic medication
↳Very powerful enzyme that
converts ketone to correct amine.
Transaminase
o Outperforms
chemical process
Screened transaminases
Biocatalytic cascade to islatravir
Nine-enzyme cascade to produce HIV drug islatravir
o No purification of intermediates was necessary
o Immobilized enzymes shown attached to spheres.
One pot reaction, but in different steps they put different enzymes
3
, Advance biocatalysis – JJ 24
Molunupiravir
Molecule active helping patients to survive COVID-19
Cascade reaction
Enzyme catalysis
- Study chemical process
- Mathematical skills so study kinetics
o Most important
o Increase rate
▪ Transition state
stabilization
- Physics, understanding the forces
- Biology, because complicated
structures activate by..
➢ Increase rate of reaction
o Transition state stabilization
➢ Do not change the equilibrium point of reactions
o Cascade to overcome equilibrium issues
➢ Specific for their substrate, active-site geometry, electrostatics
o Hydrophobic interactions (van der Waals)
o Polar (H-bonds)
o Ionic interactions (salt bridges)
o Induced fit
4
