Industrial Biotransformations

4.4 Improvements to Enzymes by Molecular Engineering Techniques
USA) offer ready-to-use error prone PCR kits using low fidelity polymerases and optimized
buffer systems.
The diversity of an enzyme library generated by epPCR is usually calculated by correlating
the base pair substitutions introduced per gene with the amino acid exchanges
introduced per enzyme molecule, i.e., an average of 1–2 base pair substitutions usually
results in one amino acid exchange. The overall size of a variant library can subsequently
be calculated by a combinatorial algorithm, as shown in Table 4.5. This algorithm is
based on the assumption that all 19 remaining amino acids can be introduced with the
same probability at a single position (E = 19). Unfortunately, this high diversity is not
created by epPCR, because an event with two or even three base pair exchanges per
codon is highly unlikely. Under optimal conditions, one nucleotide of a given codon will
be exchanged thereby leading to just 9 (instead of 64 possible) different codons encoding
4–7 (instead of 20) different amino acids. An estimation of amino acid exchanges that
could be introduced by epPCR into four different lipase genes, two originating from B.
subtilis and two from Pseudomonads, revealed that the actual number of variants was
only 22.5 and 19.5% of the theoretical number, respectively [48]. Despite these drawbacks
many examples are known from the literature showing improvements in enzyme properties,
such as (thermo-, pH- or solvent-) stability [49–51], specific activity [52] or enantioselectivity
[53, 54] by using epPCR in the first round of directed evolution.
Tab. 4.5 Theoretical number of enzyme variants in a library obtained for
an enzyme consisting of 181 amino acids (e.g., lipase A from Bacillus
subtilis) with 1–5 amino acid exchanges per molecule.
Number of amino acid exchanges
(M)
Number of variants [a]
(N)
1 3 439
2 5 880 690
3 6 666 742 230
4 5 636 730 555 465
5 3 791 264 971 605 760
a Values calculated with E = 19 using the algorithm:
N ˆ EM X!
…X M†!M!
N = number of variants at maximum size of diversity
E = number of amino acids exchanged per position
M = total number of amino acid exchanges per enzyme molecule
X = number of amino acids per enzyme molecule
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