Industrial Biotransformations

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4.4 Improvements to Enzymes by Molecular Engineering Techniques 101
single gene gene family
5´ 3´ 5´ 3´
5´ x x 3´
5´ x 3´
5´ x
…..
3´
5´ 3´
5´ 3´
random
mutagenesis
point mutations recombination
library of mutated genes library of chimeric genes
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library of enzyme variants
3
x
…..
x
x
iteration using one
improved variant
x
x
gene
expression
high-throughput
screening
selection of improved
enzyme variants / chimers
Fig. 4.2 Schematic overview of directed evolution
experiments. (1) Variant gene libraries are generated
by in vitro random mutagenesis using nonrecombinative
(introducing point mutations) or
recombinative methods. (2) Gene libraries are
x
x
x
5´ 3´
5´ 3´
5´ 3´
…..
library of chimeric enzymes
…..
iteration using a
collection of improved
variants
cloned into expression vectors and the
corresponding biocatalyst libraries are produced
in vivo by microbial host strains. (3) Biocatalysts
showing the desired properties are identified by
high-throughput screening or selection systems.