Enzyme Engineering (pdf)

Industrial Strength Proteins
Engineering enzymes for industry
Enzymes and how they work
Roles of enzymes in industry
Optimizing enzymes for industrial
use
What is an enzyme?
Here, the enzyme brings the
substrate molecules close
together in the right orientation
for the reaction to occur.
Then, the enzyme strains the bonds of
the substrates, bringing them to the
transition state. Once the reaction is
complete, the products are released.

Active site and access tunnel
• The active site is the part of an enzyme in
which the chemical reaction takes place.
• The access tunnel connects the active site
with the surface of the enzyme.
Enzyme activity is affected by
environmental factors
Why use enzymes in industrial
processes?
• Substrate/reaction-specific
• Resource-efficient
• Energy-efficient

Nitrogen fixation
• Reduction of nitrogen gas to ammonia
• The Haber-Bosch process requires a temperature of
400 o C and pressure of 200 atm
• Or nitrogen-fixing bacteria can do it at normal
temperatures and pressures
Industrial Enzymes
• Most industrial enzymes are produced by microbes.
• When possible, these enzymes are isolated from the
cells for use.
• Genetic engineering has become an important tool to
improve industrial enzyme production.
Enzymes used in industry
• Laundry detergent (proteases/lipases)
• Baking and brewing industries (amylases)
• Dairy industry (rennin)
• Pharmaceuticals (digestive aids)

Sometimes enzymes are
immobilized for industrial use
• Enzymes are attached to a solid matrix (e.g. cellulose
fibers)
• Substrate flows into reaction vessel and is converted
to product, which flows out
• Enzymes remain attached to cellulose fibers
Benefits of enzyme immobilization
Applications of Immobilized
Enzymes
• Glucose isomerase (production of high
fructose corn syrup)
• Lactase (production of lactose-free dairy
products)

Optimizing Enzymes Function
• Parameters to optimize:
• Strategies for optimization:
–Bioprospecting
–Directed evolution
Bioprospecting
• Search for organisms living in natural
environments (especially extreme
environments) to identify unique enzymes.
Applications of enzymes
discovered by bioprospecting
• Lactase from cold-loving bacteria is used in
the dairy industry
• Taq polymerase from heat-loving bacteria is
used in PCR

Directed Evolution
• Method used to evolve enzymes with new or
expanded functions
– Site-directed mutagenesis
– DNA shuffling
Site-directed mutagenesis
• Introduces mutations into DNA at precisely
determined sites
– Learn function of specific amino acids in enzyme
– Improve enzyme function
Site-directed mutagenesis
• Vector with DNA encoding
target enzyme
• Synthetic DNA strand (with
desired base change)
• Single strand extended
• After cell division – daughter
cells screened for new gene

Enzyme modified by site-directed
mutagenesis
• Trichloropropane (TCP) is a carcinogenic compound
that is persistent in the environment
• Researchers began with an enzyme that could
degrade TCP
• They created mutants by replacing amino acids in
the enzyme’s access tunnel
• The “new and improved” enzyme was able to break
down trichloropropane (TCP) with 32 times greater
activity than the wild type enzyme
DNA Shuffling
• Recombining fragments of multiple genes
encoding the same enzyme
• Improve enzyme function
DNA Shuffling
1. Parent genes – slightly different
genes that encode same product
2. Genes chopped into random
fragments
3. Gene fragments recombined
via PCR
4. DNA shuffling results in
unique, full-length genes

Genes Modified by DNA Shuffling
• Glyphosphate – active ingredient in Roundup
• Bacteria were screened for the ability to detoxify glyphosphate
• The relevant genes were isolated
• DNA shuffling was used to increase glyphosphate tolerance by
FOUR ORDERS OF MAGNITUDE
• The “new and improved” enzyme was used to develop
Roundup resistant crops
Review Questions
• Terms: enzyme, activation energy, active site, access
tunnel, specificity, substrate, product, immobilized
enzyme
• What advantages do enzymes have over live cells for
industrial applications?
• What benefits do immobilized enzymes offer over
free enzymes for use in industrial processes?
• What is bioprospecting?
• Distinguish between DNA shuffling and site-directed
mutagenesis.