Libraries of Mutants

Displayed enzyme Size (kD) Fusion protein Display format Display level (enz/phage) References 

The objective of phage display technology is the selection of clones of interest from a library of mutants. The diversity of the library will be the number of individual clones present in the collection whereas its quality will be related to the method used for its creation and to the functional diversity, i.e. the diversity from which all the clones that are unstable, misfolded or degraded have been removed. A good library will feature a high diversity and a high quality. 

The quality of a library often depends on the protocol used for its creation. For example, if a high number of cycles of error-prone PCR is performed, each mutant generated in the library will have a high number of mutations. Therefore, such a library will contain a high proportion of clones that do not fold properly. In another example, if a degenerated decapeptide is inserted in a surface loop of an enzyme, a large proportion of the library will probably be degraded by proteolysis. Hence, the choice of the method and of the experimental protocol is a crucial step. 

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In a simplified version of ISM, each site is considered only once (Figure 2.4). This means that, in the given case of four sites, 65 libraries of mutants are possible, the overall evolutionary process being convergent. Of course, not all branches have to be explored. Indeed, this embodiment of ISM has been shown to be highly successful in the rapid evolution of enantioselectivity [23,26,27] and thermostability [28]. 

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],...

All the five libraries of mutants were then tested in the hydrolysis of substrates (1) and (4—13). It turned out that most of the hits originate from libraries A or D. Figure 2.12 summarizes the relative rates of the WT- and mutant-catalyzed hydrolyses, the eight best mutants being featured. 

Several libraries of mutant ANEHs were prepared by applying epPCR at various mutation rates and transforming into E. coli BL21 (DE3). About 20 000 clones were screened, the most selective ANEH variant showing a selectivity factor of only E= 10.8 in the kinetic resolution of rac-19 [58]. Thus, this enzyme appeared to be difficult to evolve. 

Parallel to these efforts, an efficient and reliable expression system for ANEH was developed (95). Subsequently, two libraries of mutants were generated by epPCR, comprising 3500 and 4 600 clones, respectively. Mutants were discovered displaying an expression level 3.4 times higher than the original WT and a 3.3-fold enhancement of catalytic activity as measured by the hydrolysis of 4-(p-nitrophenoxy)-1,2-epoxybutane (95). The distribution of ANEH activities from the clones of these two libraries is shown in Fig. 21. 

So far, only a few groups have reported the application of this technology to libraries. As yet, suicide substrates have been applied for the selection of / -lactamase activity within libraries of mutants containing penicillin-binding proteins [6] and to... 

Enzyme engineering will increasingly drive the development of robust enzymes tailored to specific bioprocesses. The strategies used for improving the stability of enzymes will continue to focus on the generation and screening of libraries of mutants produced by random or targeted amino acid substitutions, however, alternative approaches also include... 

This strategy has also been applied for the selection of active //-lactamases from a library of mutants also containing penicillin-binding proteins. For this purpose, the protocol had to be modified to circumvent a difficulty of selections with suicide substrates in mechanisms involving a covalent intermediate. If inhibition arises from a covalent intermediate (Y in Scheme 5.2, an acyl-enzyme in the case of serine //-lactamases), enzymes whose rate of release of this intermediate (hydrolysis of the acyl-enzyme) is slow will be efficiently selected as the efficiency of inhibition depends on the ratio of rate constants k4/k3 (Scheme 5.2). To prevent the selection of enzymes with inadequate turnover, a counter-selection step was included in the protocol the library of mutants was incubated with substrate in order to block them as covalent intermediates before adding the biotinylated inhibitor. The library could be enriched from 6 ppm to 25 % active //-lactamases in four rounds of selection [62]. 

It should be mentioned that most natural aldolase enzymes can also be assayed using enzyme-coupled systems relaying the reaction to a redox process with NAD. The formation of NADH by active microbial colonies in expression libraries of mutant enzymes was detected colorimetrically in agar plates using phenazine methosulfate and nitroblue tetrazolium, which forms an insoluble precipitate. The assay was used by Williams et al. [14] and Woodhall et al. [15] for evolving sialic acid aldolases to accept non-natural aldehyde acceptors. 

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