Lipase mutant

Fig. 18. The oxyanions originating from rac-1 in the lipase-mutant J. Left (5)-l as substrate right (R)-l as substrate, showing the different degree of additional stabilization by a histidine residue (24,127).

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

Arabidopsis lipase mutant sdpT) Coexpression of Arabidopsis WRIl and DCATl 5-8 (roots, stems and leaves) 1 7 (roots with 3% w/v] exogenous sucrose) Kelly etal. (2013)... 

The lipase (PAL) used in these studies is a hydrolase having the usual catalytic triad composed of aspartate, histidine, and serine [42] (Figure 2.6). Stereoselectivity is determined in the first step, which involves the formation of the oxyanion. Unfortunately, X-ray structural characterization of the (S)- and (J )-selective mutants are not available. However, consideration of the crystal structure of the WT lipase [42] is in itself illuminating. Surprisingly, it turned out that many of the mutants have amino acid exchanges remote from the active site [8,22,40]. 

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],...

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Figure 3.5 Michael addition catalyzed by the SerlOSAla C. antarctica lipase B mutant.

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. 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

Figure 4 Course of the catalytic of the (7 )- and (S)-ester (45) as a function of time, (a) Wildtype lipase from P. aeruginosa, (b) improved mutant in the first generation.76...

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... 

Fig. 6. Typical butyrin test for lipase activity in which the agar plate is placed on a black background for visualization. White dots represent bacterial colonies those having no (even) black background contain inactive mutants (66d).

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

The infrared radiation caused by the heat of reaction of an enantioselective enzyme-catalyzed transformation can be detected by modern photovoltaic infrared (IT)-thermographic cameras equipped with focal-plane array detectors. Specifically, in the lipase-catalyzed enantioselective acylation of racemic 1-phenylethanol (20), the (K)- and (S)-substrates were allowed to react separately in the wells of microtiter plates, the (7 )-alcohol showing hot spots in the IR-thermographic images (113,114). Thus, enantioselective enzymes can be identified in kinetic resolution. However, quantification has not been achieved thus far by this method, which means that only those mutants can be identified which have E values larger than 100 (113-115). 

Another method for assaying the activity and stereoselectivity of enzymes at in vitro concentrations is based on surface-enhanced resonance Raman scattering (SERRS) using silver nanoparticles (116). Turnover of a substrate leads to the release of a surface targeting dye, which is detected by SERRS. In a model study, lipase-catalyzed kinetic resolution of a dye-labeled chiral ester was investigated. It is currently unclear how precise the method is when identifying mutants which lead to E values higher than 10. The assay appears to be well suited as a pre-test for activity. 

The results of these and other experiments are summarized in Fig. 17. A total of only 40 000 mutants was screened, which is actually a small number in our context. It is likely that upon exploration of larger portions of protein sequence space efficiently, even better lipase-variants can be identified. The assumption that millions of potential variants in the vast protein sequence space are highly enan-tioselective is not unfounded. 

The Bacillus subtilis lipase A (BSLA) is an unusual lipase because it lacks the so-called lid structural unit 13(3). Moreover, it is a small enzyme composed of only 181 amino acids. The initial results of an ongoing study are remarkable and illustrate the power of directed evolution 45,137). The desymmetrization of meso-, 4-diacetoxy-2-cyclopentene (15) was chosen as the model reaction, and the MS-based ee assay using an appropriately Ds-labeled substrate (Section III.C) provided a means to screen thousands of mutants. 

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. 

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