Pseudomonas aeruginosa lipase, directed evolution

Carballeira JD, Krumlinde P et al (2007) Directed evolution and axial chirality optimization of the enantioselectivity of Pseudomonas aeruginosa lipase towards the kinetic resolution of a racemic allene. Chem Commun 43 1913-1915... 

The improvement in enantioselectivity by the directed evolution of Pseudomonas aeruginosa lipase is shown in Figure 8(b).8 The combination of different mutagenesis methods (error-prone PCR and site-specific saturation mutagenesis) improved the enantioselectivity from E=l.l in wild type to E=25.8. 

Because the molecular basis of enantioselectivity is poorly understood, directed evolution seems to be an excellent choice for engineering enantioselective biocatalysts. Several impressive examples have been documented. In a classical study, Reetz and coworkers used error-prone PCR coupled with a 96-well plate based colorimetric screening method to increase the enantioselectivity of a Pseudomonas aeruginosa lipase toward 2-methyldecanoate. After several rounds of directed evolution, the enantioselectivity of the lipase increased from E = 1.04 (2% enantiomeric excess) to E = 25 (90-93% enantiomeric excess, ee) (E is the enantioselectivity factor). Using a similar approach. 

Fuji R, Nakagawa Y, Hiratake J et al. (2005) Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity. Protein Eng Des Sel 18(2) 93-101 Fukuda H, Kondo A, Noda H (2001) Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 92 406-416... 

Nakagawa Y, Hasegawa A, Hiratake J et al. (2007) Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity mechanistic implication for amide hydrolysis by serine hydrolases. Protein Eng Des Sel 20(7) 339-346 Nardini M, Lang DA, Liebeton K et al. (2000) Crystal stracture of Pseudomorms aeruginosa lipase in the open conformation. The prototype for family LI of bacterial lipases. J Biol Chem 275 31219-31225... 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

Finally, it is interesting to note that in most cases enhanced enantioselectivity was shown to be due to a reduced value of / cat/Am for the non-preferred enantiomer 143). This result is contrasted with the results of directed evolution of the lipase from Pseudomonas aeruginosa, in which case the value of for the preferred... 

Lipases are the most frequently used enzymes in organic chemistry, catalyzing the hydrolysis of carboxylic acid esters or the reverse reaction in organic solvents [3,5,34,70]. The first example of directed evolution of an enantioselective enzyme according to the principle outlined in Fig. 11.2 concerns the hydrolytic kinetic resolution of the chiral ester 9 catalyzed by the bacterial lipase from Pseudomonas aeruginosa [8], This enzyme is composed of 285 amino acids [32]. It is an active catalyst for the model reaction, but enantioselectivity is poor (ee 5 % in favor of the (S)-acid 10 at about 50 % conversion) (Fig. 11.10) [71]. The selectivity factor E, which reflects the relative rate of the reactions of the (S)- and (R)-substrates, is only 1.1. 

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... 

Figure 10.10 Reaction catalysed by a lipase from Pseudomonas aeruginosa chosen for directed evolution of enantioselectivity.

In the following case, we shall look at the molecular breeding of the lipase from the bacterium Pseudomonas aeruginosa. This particular molecular breeding experiment was the first example of the directed evolution of an enantioselective enzyme. The wild-type enzyme catalyses the hydrolysis of esters to carboxylic acid (Figure 10.10),and shows very little enantioselectivity-only two per cent enantiomeric excess biased towards the (S) configuration. 

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