Lipases, from Pseudomonas aeruginosa

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. 

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],...

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... 

Some l-(2-furyl)-l-alkanols have also been resolved by hydrolase-catalyzed acylations (Scheme 4.20). Thus l-(2-furyl)-l-ethanol (46) is efficiently resolved by acylation with vinyl acetate catalyzed either by Lipozyme IM or PPL [77]. Resolution with a more complex acyl donor, ethoxyvinyl methyl fumarate, catalyzed by Lipase LIP (from Pseudomonas aeruginosa) has also been achieved [95]. The... 

Hydrolase-catalyzed domino reactions incorporating a resolution and a subsequent cycloaddition reaction have been described [95-97]. This constitutes an attractive approach to complex synthetic intermediates. For example, the l-(3-methyl-2-furyl)]propanol roc-93 reacts with ethoxyvinyl methyl fumarate (94) catalyzed by Lipase LIP (from Pseudomonas aeruginosa) to furnish a dienophilic fumarate ester, which spontaneously undergoes an intramolecular Diels-Alder reaction with the furan moiety furnishing exclusively the syn-adduct, the oxabicy-clohexene 95 in excellent along with the remaining alcohol S-96 (Scheme 4.31) [95]. A similar approach has been used for a procedure that includes a series of domino reactions that includes dynamic kinetic resolution of the 3-vinylcyclohex-... 

Conventional gas chromatography (GC) based on the use of chiral stationary phases can handle only a few dozen ee determinations per day. In some instances GC can be modified so that, in optimal situations, about 700 exact ee and E determinations are possible per day [29]. Such meclium-throughputmay suffice in certain applications. The example concerns the lipase-catalyzed kinetic resolution of the chiral alcohol (R)- and (S)-18 with formation of the acylated forms (R)- and (S )-19. Thousands of mutants of the lipase from Pseudomonas aeruginosa were created by error-prone PCR for use as catalysts in the model reaction and were then screened for enantioselectivity [29]. 

Nadkarni, S.R. 1971. Studies on bacterial lipase. Part II. Study of the characteristics of partially purified lipase from Pseudomonas aeruginosa. Enzymologia 40, 302—313. 

Hundreds of impressive examples of enantioselective lipase-catalysed reactions are known, including industrial processes as in the case of the BASF method of chiral amine production (Collins et al. 1997 Breuer et al. 2004 Schmid and Verger 1998). However, the classical problem of substrate acceptance or lack of enantioselectivity (or both) persists. We were able to meet this challenge in model studies regarding the hydrolytic kinetic resolution of the ester rac-1 with formation of carboxylic acid 2, catalysed by the lipase from Pseudomonas aeruginosa. The wild-type (WT) lipase is only slightly (S )-selective, the selectivity factor amounting to a mere E = 1.1 (Scheme 1). 

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. 

In some samples of the wild-type lipase from Pseudomonas aeruginosa we have observed small variations in the ee... 

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... 

In a classical study the lipase-catalysed enantioselective hydrolysis of racemic p-nitrophenyl-2-methyldecanoate was chosen as the test reaction [15] (Fig. 8). The p-nitrophenyl ester was employed in the kinetic resolution instead of the methyl or ethyl ester, in order to make screening possible [76] (see below). The lipase from the bacterium Pseudomonas aeruginosa PAOl [77] was used as the enzyme [ 15]. The wild-type enzyme shows an enantioselectivity (ee) of only 2 % in favour of the (S)-configured 2-methyldecanoic acid, which means that the enzyme had essentially no preference for either of the enantiomeric forms. 

The enantioselective ROP of 3-methyl-4-oxa-6-hexanolide (MOHEL) was catalyzed in bulk at 60 °C [34]. A comparison of the initial rate of poly(MOHEL) formation from the (R) and (S) antipodes showed that the (S) enantiomer had an initial rate that was seven times larger. Lipase from Pseudomonas aeruginosa and Pseudomonas fluorescens catalyzed the polymerization of (S)-MOHEL but not (R)-MOHEL (Scheme 4.18). 

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. ... 

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. 

Misset O, Gerritse G, Jaeger KE et al. (1994) The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis. Protein Eng 7(4) 523-529 Murahdhar RV, ChirumanuUa RR, Marchant R et al. (2002) Understanding lipase stereoselectivity. World 1 Microbiol Biotechnol 18 81-97... 

Sharon C, Furugoh S, Yamakido T et al. (1998) Purification and characterization of a lipase from Pseudomonas aeruginosa KKA-5 and its role in ctistor oil hydrolysis. J Ind Microbiol Biotech-nol 20 304-307... 

Devappa RK, Makkar HPS, Becker K (2010) Optimization of conditions for the extraction of phoibol esters from Jatropha oil. Biomass Bioenerg 34 1125-1133 Dharmsthiti S, Kuhasuntisuk B (1998) Lipase from Pseudomonas aeruginosa LP602 biochemical properties and application for wastewater treatment. J Ind Microbiol Biotechnol... 

---