Mutagenesis epPCR

Systematic experimental and theoretical studies of this kind are helpful in performing in vitro evolution of enantioselectivity. Nevertheless, several questions are not fully answered. Are remote mutations more important than those close to the active site, or is the opposite true Is it more effective to allow randomization all over the enzyme rather than focusing on the region around the active site (or vice versa) To be sure, when applying epPCR or any other mutagenesis method that more or less... 

Following epPCR and saturation mutagenesis at hot spots, the o-selective hydan-toinase from Arthrobacter sp. DSM 9771 was converted into L-selective variants. The best L-selective mutant showed a value of 20% ee at about 30% conversion, compared to the WT displaying ee = 40% in favor of the D-methionine-derivative. With the help of an appropriate L-carbamoylase, L-methionine itself was produced. This academic/ industrial effort provided several selective hydantoinases of industrial interest (O May, (Degussa-Hiils), personal communication, 2005). 

In 1989, a very practical mutagenesis method was described by Leung et al. 39), which was later improved by Cadwell and Joyce 40). Error-prone polymerase chain reaction (epPCR), as it is called, is based on the classical DNA amplification... 

If the slopes of the absorption/time curves differ considerably, a positive hit is indicated (i.e., an enantioselective lipase-variant has been identified) (16). Figure 5 shows two typical experimental plots, illustrating the presence of a non-selective lipase (top) and a hit (bottom) (16). As a consequence of the crudeness of the test, quantitative evaluation is not possible. Therefore, the hits need to be investigated separately in laboratory-scale reactions and evaluated quantitatively by conventional chiral GC. About 800 plots of this kind can easily be recorded per day. A total of 40 000 lipase-variants were generated by epPCR, saturation mutagenesis, cassette mutagenesis, and DNA shuffling and screened in the model reaction. 

It is thus dear that the combination of epPCR and saturation mutagenesis constitutes an efficient way to explore protein sequence space with respect to enantioselectivity. Indeed, this strategy led to the creation of several other highly (S)-selective variants (ee = 88 - 91 % E= 20 - 25) [9,42], all of them being the descendents of the parent wild-type lipase, which is only slightly (S)-selective. 

The wild-type enzyme leads to an ee value of only 38 % in favor of (R,S)-12. Following an initial round of epPCR-based random mutagenesis, a variant showing an ee value of 60 % was identified. This was increased to about 70 % ee in the second round. Current... 

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