Specificity directed evolution

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

A more recent study focused on the directed evolution of the co-transaminase from Vibrio fiuvialis JS17, specifically with the aim to eliminate product inhibition by aliphatic ketones while maintaining high enantioselectivity. This was achieved by screening 85 000 clones produced by epPCR [72]. 

Several other enzymes have been investigated by directed evolution in order to control substrate specificity, although most of the studies are at an early stage. These... 

Lee, PC. et al., Alteration of product specificity of Aeropyrum pemix famesylgeranyl diphosphate synthase (fgs) by directed evolution. Protein Eng. Design Sel. 17, 771, 2004. 

Hall, B.G. (1981) Changes in the substrate specificities of an enzyme during directed evolution of new functions. 

Figure 6.1 Alteration of substrate specificity of sialic acid aldolase by directed evolution...

Carr, R., Alexeeva, M., Enright, A., Eve, T.S.C., Dawson, M.J. and Turner, N.J., Directed evolution of an amine oxidase possessing both broad substrate specificity and high enantios-electivity. Angew. Chem. Int. Ed., 2003, 42, 4807. 

H.M. Cohen, D.S. Tawfik, and A.D. Griffiths Altering the Sequence Specificity of Haeiii Methyltransferase by Directed Evolution Using In Vitro Compartmentalisation. Prot. Eng. Des. Sel. 17, 3 (2004). 

Scheme 2.32 Directed evolution to broaden the substrate specificity of MAO-N.

Several other UV/Vis-based ee assays have been developed, but their general application in directed evolution remains to be demonstrated 66-70). One of them is a well- designed screening system based on enzyme immunoassays (85). The success of the ee assay depends upon the availability of specific antibodies that are easily raised for almost any chiral product of interest. In a given reaction, two antibodies are needed, one that measures the product concentration and the other that measures the amount of one of the enantiomers. About 1000 ee determinations are possible per day, the precision amounting to + 9% (85). 

Thus, because the test can be carried out on 96-well microtiter plates, high throughput is possible. Of course, the inherent disadvantage noted above for some of the colorimetric tests also applies here, namely, the fact that the optimization of a potential catalyst is focused on a specific substrate 11 modified by incorporation of a probe, in this case the fluorogenic moiety 14. However, one can expect the test to be useful in directed evolution projects in which proof of principle is the goal. Moreover, this kind of approach can be used in very practical applications, namely as a pre-test for the activity of enzymes. 

In the sulfoxidation, small to appreciable amounts of over oxidation with formation of undesired sulfone were observed, a result that implies that kinetic resolution may be involved in influencing the overall stereochemical result 105). This was shown to be the case. Indeed, some of the mutants are also excellent catalysts in the kinetic resolution of racemic sulfoxides such as 25 105). Directed evolution was then applied successfully to eliminate undesired sulfone formation, specifically, by going through a second cycle of epPCR 105). This is significant because it shows for the first time that an undesired side reaction can be eliminated by directed evolution. 

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