Enzyme Engineering (pdf)
Enzyme Engineering (pdf)
Enzyme Engineering (pdf)
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Industrial Strength Proteins<br />
<strong>Engineering</strong> enzymes for industry<br />
<strong>Enzyme</strong>s and how they work<br />
Roles of enzymes in industry<br />
Optimizing enzymes for industrial<br />
use<br />
What is an enzyme?<br />
Here, the enzyme brings the<br />
substrate molecules close<br />
together in the right orientation<br />
for the reaction to occur.<br />
Then, the enzyme strains the bonds of<br />
the substrates, bringing them to the<br />
transition state. Once the reaction is<br />
complete, the products are released.
Active site and access tunnel<br />
• The active site is the part of an enzyme in<br />
which the chemical reaction takes place.<br />
• The access tunnel connects the active site<br />
with the surface of the enzyme.<br />
<strong>Enzyme</strong> activity is affected by<br />
environmental factors<br />
Why use enzymes in industrial<br />
processes?<br />
• Substrate/reaction-specific<br />
• Resource-efficient<br />
• Energy-efficient
Nitrogen fixation<br />
• Reduction of nitrogen gas to ammonia<br />
• The Haber-Bosch process requires a temperature of<br />
400 o C and pressure of 200 atm<br />
• Or nitrogen-fixing bacteria can do it at normal<br />
temperatures and pressures<br />
Industrial <strong>Enzyme</strong>s<br />
• Most industrial enzymes are produced by microbes.<br />
• When possible, these enzymes are isolated from the<br />
cells for use.<br />
• Genetic engineering has become an important tool to<br />
improve industrial enzyme production.<br />
<strong>Enzyme</strong>s used in industry<br />
• Laundry detergent (proteases/lipases)<br />
• Baking and brewing industries (amylases)<br />
• Dairy industry (rennin)<br />
• Pharmaceuticals (digestive aids)
Sometimes enzymes are<br />
immobilized for industrial use<br />
• <strong>Enzyme</strong>s are attached to a solid matrix (e.g. cellulose<br />
fibers)<br />
• Substrate flows into reaction vessel and is converted<br />
to product, which flows out<br />
• <strong>Enzyme</strong>s remain attached to cellulose fibers<br />
Benefits of enzyme immobilization<br />
Applications of Immobilized<br />
<strong>Enzyme</strong>s<br />
• Glucose isomerase (production of high<br />
fructose corn syrup)<br />
• Lactase (production of lactose-free dairy<br />
products)
Optimizing <strong>Enzyme</strong>s Function<br />
• Parameters to optimize:<br />
• Strategies for optimization:<br />
–Bioprospecting<br />
–Directed evolution<br />
Bioprospecting<br />
• Search for organisms living in natural<br />
environments (especially extreme<br />
environments) to identify unique enzymes.<br />
Applications of enzymes<br />
discovered by bioprospecting<br />
• Lactase from cold-loving bacteria is used in<br />
the dairy industry<br />
• Taq polymerase from heat-loving bacteria is<br />
used in PCR
Directed Evolution<br />
• Method used to evolve enzymes with new or<br />
expanded functions<br />
– Site-directed mutagenesis<br />
– DNA shuffling<br />
Site-directed mutagenesis<br />
• Introduces mutations into DNA at precisely<br />
determined sites<br />
– Learn function of specific amino acids in enzyme<br />
– Improve enzyme function<br />
Site-directed mutagenesis<br />
• Vector with DNA encoding<br />
target enzyme<br />
• Synthetic DNA strand (with<br />
desired base change)<br />
• Single strand extended<br />
• After cell division – daughter<br />
cells screened for new gene
<strong>Enzyme</strong> modified by site-directed<br />
mutagenesis<br />
• Trichloropropane (TCP) is a carcinogenic compound<br />
that is persistent in the environment<br />
• Researchers began with an enzyme that could<br />
degrade TCP<br />
• They created mutants by replacing amino acids in<br />
the enzyme’s access tunnel<br />
• The “new and improved” enzyme was able to break<br />
down trichloropropane (TCP) with 32 times greater<br />
activity than the wild type enzyme<br />
DNA Shuffling<br />
• Recombining fragments of multiple genes<br />
encoding the same enzyme<br />
• Improve enzyme function<br />
DNA Shuffling<br />
1. Parent genes – slightly different<br />
genes that encode same product<br />
2. Genes chopped into random<br />
fragments<br />
3. Gene fragments recombined<br />
via PCR<br />
4. DNA shuffling results in<br />
unique, full-length genes
Genes Modified by DNA Shuffling<br />
• Glyphosphate – active ingredient in Roundup<br />
• Bacteria were screened for the ability to detoxify glyphosphate<br />
• The relevant genes were isolated<br />
• DNA shuffling was used to increase glyphosphate tolerance by<br />
FOUR ORDERS OF MAGNITUDE<br />
• The “new and improved” enzyme was used to develop<br />
Roundup resistant crops<br />
Review Questions<br />
• Terms: enzyme, activation energy, active site, access<br />
tunnel, specificity, substrate, product, immobilized<br />
enzyme<br />
• What advantages do enzymes have over live cells for<br />
industrial applications?<br />
• What benefits do immobilized enzymes offer over<br />
free enzymes for use in industrial processes?<br />
• What is bioprospecting?<br />
• Distinguish between DNA shuffling and site-directed<br />
mutagenesis.