Pseudomonas aeruginosa, lipase

Pseudomonas aeruginosa lipase-catalyzed hydrolysis of racemic ester 23 proceeds with very low enantioselectivity E = 1.1). Sequential use of error-prone PCR, saturation mutagenesis at chosen spots and DNA shuffling resulted in the formation of a mutant whose enantioselectivity was over 50. 

The low-temperature method was then applied to the resolution of ( )-2-hydroxy-2-(pentafluorophenyl)acetonitrile (7) (Fig. which is usahle for the syntheses of a variety of ethane diols, amino alcohols containing CgFj groups as novel chiral ligands. After screening lipases such as Amano PS and AK, lipase LIP Pseudomonas aeruginosa lipase immobilized on Hyflo Super-Cel, Toyobo,... 

Pseudomonas aeruginosa lipase (a crude solution in acetone)... 

The WT lipase leads to an ee value of only 38% in favor of the (If ,45) enantiomer. The application of low-error epPCR increased the enantioselectivity slightly, but high-error rate epPCR turned out to be more successful, with several mutants showing ee values of 54-58% (45,137). The results are in line with the experience gained in the Pseudomonas aeruginosa lipase project (Section IV.A. 1). Of course, a library produced by high mutation rate can also contain hits that have only one amino acid exchange, and this was indeed observed in several cases. 

The sterically rather demanding racemic binaphthylalkylamines 126 and 127 can be efficiently resolved in diisopropyl ether using Pseudomonas aeruginosa lipase (LIP) as catalyst and trifluoroethyl butyrate as acylating agent for 126 and ethyl butyrate for 127 (Scheme 4.38) [125]. 

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. 

Table 11.1-14. Lipase-catalyzed enantiomer-differentiating hydrolysis of esters of racemic primary alcohols in aqueous solution (PPL pig pancreas lipase, PCL Pseudomonas cepacia lipase, PCL-A Pseudomonas cepacia lipase, Sumitomo, PSL Pseudomonas sp. lipase, PAL Pseudomonas aeruginosa lipase, HLL Humicola lanuginosa lipase, CAL-B Candida antarctica B lipase, CRL Candida rugosa lipase).

The Pseudomonas aeruginosa lipase (immobilised on hyflo Super-Cel) catalysed kinetic resolution of (rac)-2-(acetyloxy)-2-(pentafluorophenyl)acetonitrile gave en-antiomerically pure cyanohydrin and its antipodal ester [142-144l... 

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... 

Scheme 2.47 Screening for Pseudomonas aeruginosa lipase mutants showing enhanced enan-tioselectivities using a chromogenic surrogate substrate...

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