Lipase screening tests

Lipase-catalyzed conversion of FAStEs to sterols in the presence of MeOH was applied to astaxanthin FA esters to free astaxanthin, because a screening test showed that commercial lipases did not hydrolyze astaxanthin FA esters. A mixture of astaxanthin and its mono- and diesters (5 80 15, by mol) was treated in the presence of 50wt% water with 5 mol EtOH for FAs in astaxanthin esters using P. aeruginosa lipase. After the reaction, the molar ratio of astaxanthin and its mono- and diesters was 89 10 1, showing that the lipase efficiently converts astaxanthin esters to free astaxanthin. The free form was also purified by n-hexane fractionation with 69% recovery of the initial content of astaxanthin in the mixture of its free and ester forms (Nagao et al., 2003). 

To test the selectivity of the self-screening process, six other enzymes belonging to the hydrolase family were tested under the same set of conditions as in DCL-A. These enzymes were butyrylcholinesterase, horse fiver esterase (HLE, EC 3.1.1.1), Candida cylindracea lipase (CCL, EC 3.1.1.3), (3-galactosidase ((3-Gal, EC 3.2.1.23), trypsin (EC 3.4.21.4), and... 

If the slopes of the absorption/time curves differ considerably, a positive hit is indicated (i.e., an enantioselective lipase-variant has been identified) (16). Figure 5 shows two typical experimental plots, illustrating the presence of a non-selective lipase (top) and a hit (bottom) (16). As a consequence of the crudeness of the test, quantitative evaluation is not possible. Therefore, the hits need to be investigated separately in laboratory-scale reactions and evaluated quantitatively by conventional chiral GC. About 800 plots of this kind can easily be recorded per day. A total of 40 000 lipase-variants were generated by epPCR, saturation mutagenesis, cassette mutagenesis, and DNA shuffling and screened in the model reaction. 

Fig. 4.4 Phenotype of 4-hydroxybutyrate dehydrogenase-positive (A) and lipase/esterase-positive (B) E. coli clones. A, 4-Hydroxy-butyrate dehydrogenase-positive clones are marked by arrows. Tetrazolium indicator plates [37] containing 4-hydroxybutyrate as test substrate were employed for the screening procedure [9], Positive clones were identified by formation of a deep-red formazan inside the colonies. B, Lipase/esterase activity of the clones was detected on LB agar [32] containing tributyrin as test substrate [14]. Zones of clearance around the colonies were indicative for lipase/ esterase activity.

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. 

Typical commercial enzymes reported for resolution of amino acids were tested. Whole cell systems containing hydantoinase were found to produce only a-monosubstituted amino acids" the acylase-catalyzed resolution of Xacyl amino acids had extremely low rates toward a-dialkylated amino acids and the nitrilase system obtained from Novo Nordisk showed no activity toward the corresponding 2-amino-2-ethylhexanoic amide. Finally, a large-scale screening of hydrolytic enzymes for enantioselective hydrolysis of racemic amino esters was carried out. Of all the enzymes and microorganisms screened, pig hver esterase (PLE) and Humicola langinosa lipase (Lipase CE, Amano) were the only ones found to catalyze the hydrolysis of the substrate (Scheme 9.6). 

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