Lipase from Pseudomonas aeruginosa PAL

Finally, the region of accessible protein sequence space was extended by developing a modified version of Stemmer s combinatorial multiple-cassette mutagenesis (CMCM) 

These initial systematic studies regarding the directed evolution of PAL allowed several conclusions to be made. Protein sequence space can be explored successfully by applying the following strategies [8c,33j  

Efforts were also made to invert the sense of enantioselectivity in the hydrolytic kinetic resolution of ester (1) using PAL with preferential formation of (R)-2 [40,411-Using epPCR and DNA shuffling, an (R)-selective mutant showing an E value of 30 was evolved by screening about 45 000 clones for the (R) enantiomer. The best mutant is characterized by 11 mutations, which are different from those of the best (S)-selective variant X [41]. 

In order to approximate the activity of the best variant X, the supernatants were used to perform kinetics ]8c]. The kcat values of variant X turned out to be 9.6 s for (S)-l and 1.2 s for (i )-l. In going from the WT to variant X, the kcat/Km value (Imoff s ) increased significantly for (S)-l, kcat/lQn = 9.0x 10 (wild type) and 3.7x10 (variant X) for (i )-l, kcat/l m= 3.5x 10 (wild type) and 8.4x10 (variant X). Thus, although these are only rough numbers, they show that variant X is much more active than the WT. 

The initial results of the MM/QM study regarding the source of enhanced enantioselectivity led to several plausible conclusions ]36a[. First, only two of the six amino add substitutions of mutant X influence enantioselectivity substantially. 

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Figure 2.11 CASTing of the lipase from Pseudomonas aeruginosa (PAL) leading to the construction of five libraries of mutants (A-E) produced by simultaneous randomization at sites composed of two amino acids. (For illustrative purposes, the binding of substrate (1) is shown) [25],...

A second example of the use of directed molecular evolution for natural product synthesis is the use of lipases by Reetz and colleagues. This work is based on the kinetic hydrolytic resolution of racemic mixtures, in which one enantiomer is preferentially hydrolyzed and the chiral product is thus enriched. Utilizing both random mutagenesis and directed techniques such as CAST,64 they have improved the stereoselectivity of a lipase from Pseudomonas aeruginosa (PAL) on a number of occasions with different substrates. One of the first examples utilized the model substrate 2-methyldecanoic acid /xnitrophenyl ester, for which the wild-type enzyme has an enantioselectivity of E= 1.1. As a consequence of five mutations accumulated through random mutagenesis, followed by saturation mutagenesis, the enantioselectivity was increased to 25.8.123 More... 

The lipase from Pseudomonas aeruginosa (PAL) catalyzes the hydrolysis of 2-me-thyldecanoic acid p-nitrophenyl ester with only 2% ee in favor of the (S)-acid. Reetz and Jaeger used four rounds of error-prone PCR and screening on enantiomerically pure R and S substrates to generate a more enantioselective variant that produced the desired (S)-acid with 81% eell57l Additional cycles of error-prone PCR in combination with saturation mutagenesis further improved the enantioselectivity of this enzyme, which hydrolyzes the 2-methyldecanoic acid p-nitrophenyl ester with 91 % ee (E = 25.8) in favor of the (S)-acid 1Z. ... 

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