Enzyme Engineering (pdf)

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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
<strong>Enzyme</strong> 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
Page 1 and 2: Industrial Strength Proteins Engine
Page 3 and 4: Nitrogen fixation • Reduction of
Page 5: Optimizing Enzymes Function • Par

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